*AI Summary*
*# *I. Analyze and Adopt**
*Domain:* Molecular Virology and Viral Genetics
*Expert Persona:* Senior Research Scientist in Molecular Virology
*Vocabulary/Tone:* Academic, mechanistic, precise, and focused on biochemical pathways and evolutionary implications.
---
### *II. Reviewing Group*
The ideal group to review this material would be *Graduate Students in Biomedical Sciences and Research Fellows in Pathogenesis.* These individuals are focused on the molecular "rules of the game" that dictate how viral pathogens replicate and evolve.
---
### *III. Synthesis and Summary*
*Abstract:*
This technical lecture details the fundamental mechanisms of RNA-dependent RNA synthesis across various viral families. Because host cells lack the machinery to replicate RNA from an RNA template, all RNA viruses (excluding retroviruses) must encode an RNA-dependent RNA polymerase (RdRp). The discussion covers the biochemical basis of RdRp catalysis—specifically the "two-metal" mechanism coordinated by aspartate residues—and the structural "right-hand" motif common to these enzymes. Distinct replication strategies are analyzed: plus-strand viruses (e.g., Polio) utilize protein-priming and circularization; minus-strand viruses (e.g., Influenza, VSV) employ "cap-snatching" or "slipping" for polyadenylation; and double-stranded RNA viruses (e.g., Reovirus) transcribe mRNA within the viral capsid to evade host sensors. The session concludes with an analysis of viral evolution, highlighting high mutation rates due to the lack of proofreading (excepting the Coronaviridae exonuclease) and the role of template-switching in recombination.
*Key Takeaways and Technical Summary:*
* *0:13 – Historical Context and RNA as Genetic Material:* Evolution of virology from the crystallization of Tobacco Mosaic Virus (TMV) to the 1956 Frankel-Conrad experiment confirming RNA as a genetic carrier, necessitating the study of non-canonical replication.
* *3:59 – The Baltimore Scheme & RdRp Location:* Different viral classes manage RdRp differently:
* *Negative-strand and dsRNA viruses* must carry the RdRp within the virion because their genomes cannot be immediately translated.
* *Plus-strand viruses* do not carry the enzyme, as their genome serves directly as mRNA for initial translation.
* *11:14 – Higher-Order RNA Structure:* RNA genomes are not linear strings but complex 3D structures (stem-loops, pseudo-knots) that facilitate protein binding and replication initiation.
* *14:07 – Universal Rules of Synthesis:* RNA is synthesized in a 5’ to 3’ direction while the template is read 3’ to 5’. Initiation can be *de novo* or primer-dependent (protein or capped primers).
* *17:19 – Biochemical Mechanism of Catalysis:* RdRps utilize a two-metal (Magnesium) mechanism. Two conserved aspartate residues coordinate these ions to facilitate a nucleophilic attack on incoming NTPs, releasing pyrophosphate.
* *23:15 – Structural Conservation (The "Right Hand"):* Polymerases share a conserved structure resembling a right hand with "palm" (active site), "fingers," and "thumb" domains. Polio RdRp features a "closed" conformation where fingers and thumb interact.
* *31:36 – Polio Virus (Picornaviridae) Strategy:* Utilizes a protein primer (VPg) uridylated at a cis-acting RNA element (CRE). Replication requires genome circularization mediated by host poly-A binding proteins.
* *40:30 – Subgenomic mRNAs (Alpha and Coronaviridae):* These viruses produce mRNAs shorter than the genome. Coronaviruses utilize a unique "template switching" mechanism where the polymerase jumps to a leader sequence, facilitating high rates of recombination.
* *45:02 – The "Switch" in Negative-Strand Viruses:* For VSV and Influenza, the concentration of nucleocapsid (N) protein dictates whether the RdRp produces short, capped mRNAs or full-length genomic copies.
* *50:36 – Influenza (Orthomyxoviridae) Specifics:* Occurs in the nucleus. Uses "cap-snatching" (stealing 5' caps from host pre-mRNA) as primers. Polyadenylation occurs via "slipping" when the RdRp hits a stretch of U residues and cannot move forward due to steric hindrance.
* *55:52 – Reovirus (dsRNA) Sequestration:* Synthesis occurs entirely within the viral core to evade host cytoplasmic RNA sensors. mRNA is extruded through turrets located at the icosahedral vertices.
* *59:52 – Fidelity and Evolution:* RNA polymerases lack proofreading, leading to high mutation rates (1 in 10,000 bases). Coronaviruses are the exception, encoding an exonuclease (ExoN) that allows for much larger genomes (up to 40kb) by correcting errors.
* *1:04:46 – Recombination Risks:* High-frequency recombination (template switching) is a driver of viral diversity and can compromise the stability of live-attenuated vaccines, such as the oral polio vaccine, in the human gut.
AI-generated summary created with gemini-3-flash-preview for free via RocketRecap-dot-com. (Input: 29,527 tokens, Output: 1,127 tokens, Est. cost: $0.0181).Below, I will provide input for an example video (comprising of title, description, and transcript, in this order) and the corresponding abstract and summary I expect. Afterward, I will provide a new transcript that I want a summarization in the same format.
**Please give an abstract of the transcript and then summarize the transcript in a self-contained bullet list format.** Include starting timestamps, important details and key takeaways.
Example Input:
Fluidigm Polaris Part 2- illuminator and camera
mikeselectricstuff
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Fluidigm Polaris part 1 : • Fluidigm Polaris (Part 1) - Biotech g...
Ebay listings: https://www.ebay.co.uk/usr/mikeselect...
Merch https://mikeselectricstuff.creator-sp...
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mikeselectricstuff
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40 Comments
@robertwatsonbath
6 hours ago
Thanks Mike. Ooof! - with the level of bodgery going on around 15:48 I think shame would have made me do a board re spin, out of my own pocket if I had to.
1
Reply
@Muonium1
9 hours ago
The green LED looks different from the others and uses phosphor conversion because of the "green gap" problem where green InGaN emitters suffer efficiency droop at high currents. Phosphide based emitters don't start becoming efficient until around 600nm so also can't be used for high power green emitters. See the paper and plot by Matthias Auf der Maur in his 2015 paper on alloy fluctuations in InGaN as the cause of reduced external quantum efficiency at longer (green) wavelengths.
4
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1 reply
@tafsirnahian669
10 hours ago (edited)
Can this be used as an astrophotography camera?
Reply
mikeselectricstuff
·
1 reply
@mikeselectricstuff
6 hours ago
Yes, but may need a shutter to avoid light during readout
Reply
@2010craggy
11 hours ago
Narrowband filters we use in Astronomy (Astrophotography) are sided- they work best passing light in one direction so I guess the arrows on the filter frames indicate which way round to install them in the filter wheel.
1
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@vitukz
12 hours ago
A mate with Channel @extractions&ire could use it
2
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@RobertGallop
19 hours ago
That LED module says it can go up to 28 amps!!! 21 amps for 100%. You should see what it does at 20 amps!
Reply
@Prophes0r
19 hours ago
I had an "Oh SHIT!" moment when I realized that the weird trapezoidal shape of that light guide was for keystone correction of the light source.
Very clever.
6
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@OneBiOzZ
20 hours ago
given the cost of the CCD you think they could have run another PCB for it
9
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@tekvax01
21 hours ago
$20 thousand dollars per minute of run time!
1
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@tekvax01
22 hours ago
"We spared no expense!" John Hammond Jurassic Park.
*(that's why this thing costs the same as a 50-seat Greyhound Bus coach!)
Reply
@florianf4257
22 hours ago
The smearing on the image could be due to the fact that you don't use a shutter, so you see brighter stripes under bright areas of the image as you still iluminate these pixels while the sensor data ist shifted out towards the top. I experienced this effect back at university with a LN-Cooled CCD for Spectroscopy. The stripes disapeared as soon as you used the shutter instead of disabling it in the open position (but fokussing at 100ms integration time and continuous readout with a focal plane shutter isn't much fun).
12
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mikeselectricstuff
·
1 reply
@mikeselectricstuff
12 hours ago
I didn't think of that, but makes sense
2
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@douro20
22 hours ago (edited)
The red LED reminds me of one from Roithner Lasertechnik. I have a Symbol 2D scanner which uses two very bright LEDs from that company, one red and one red-orange. The red-orange is behind a lens which focuses it into an extremely narrow beam.
1
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@RicoElectrico
23 hours ago
PFG is Pulse Flush Gate according to the datasheet.
Reply
@dcallan812
23 hours ago
Very interesting. 2x
Reply
@littleboot_
1 day ago
Cool interesting device
Reply
@dav1dbone
1 day ago
I've stripped large projectors, looks similar, wonder if some of those castings are a magnesium alloy?
Reply
@kevywevvy8833
1 day ago
ironic that some of those Phlatlight modules are used in some of the cheapest disco lights.
1
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1 reply
@bill6255
1 day ago
Great vid - gets right into subject in title, its packed with information, wraps up quickly. Should get a YT award! imho
3
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@JAKOB1977
1 day ago (edited)
The whole sensor module incl. a 5 grand 50mpix sensor for 49 £.. highest bid atm
Though also a limited CCD sensor, but for the right buyer its a steal at these relative low sums.
Architecture Full Frame CCD (Square Pixels)
Total Number of Pixels 8304 (H) × 6220 (V) = 51.6 Mp
Number of Effective Pixels 8208 (H) × 6164 (V) = 50.5 Mp
Number of Active Pixels 8176 (H) × 6132 (V) = 50.1 Mp
Pixel Size 6.0 m (H) × 6.0 m (V)
Active Image Size 49.1 mm (H) × 36.8 mm (V)
61.3 mm (Diagonal),
645 1.1x Optical Format
Aspect Ratio 4:3
Horizontal Outputs 4
Saturation Signal 40.3 ke−
Output Sensitivity 31 V/e−
Quantum Efficiency
KAF−50100−CAA
KAF−50100−AAA
KAF−50100−ABA (with Lens)
22%, 22%, 16% (Peak R, G, B)
25%
62%
Read Noise (f = 18 MHz) 12.5 e−
Dark Signal (T = 60°C) 42 pA/cm2
Dark Current Doubling Temperature 5.7°C
Dynamic Range (f = 18 MHz) 70.2 dB
Estimated Linear Dynamic Range
(f = 18 MHz)
69.3 dB
Charge Transfer Efficiency
Horizontal
Vertical
0.999995
0.999999
Blooming Protection
(4 ms Exposure Time)
800X Saturation Exposure
Maximum Date Rate 18 MHz
Package Ceramic PGA
Cover Glass MAR Coated, 2 Sides or
Clear Glass
Features
• TRUESENSE Transparent Gate Electrode
for High Sensitivity
• Ultra-High Resolution
• Board Dynamic Range
• Low Noise Architecture
• Large Active Imaging Area
Applications
• Digitization
• Mapping/Aerial
• Photography
• Scientific
Thx for the tear down Mike, always a joy
Reply
@martinalooksatthings
1 day ago
15:49 that is some great bodging on of caps, they really didn't want to respin that PCB huh
8
Reply
@RhythmGamer
1 day ago
Was depressed today and then a new mike video dropped and now I’m genuinely happy to get my tear down fix
1
Reply
@dine9093
1 day ago (edited)
Did you transfrom into Mr Blobby for a moment there?
2
Reply
@NickNorton
1 day ago
Thanks Mike. Your videos are always interesting.
5
Reply
@KeritechElectronics
1 day ago
Heavy optics indeed... Spare no expense, cost no object. Splendid build quality. The CCD is a thing of beauty!
1
Reply
@YSoreil
1 day ago
The pricing on that sensor is about right, I looked in to these many years ago when they were still in production since it's the only large sensor you could actually buy. Really cool to see one in the wild.
2
Reply
@snik2pl
1 day ago
That leds look like from led projector
Reply
@vincei4252
1 day ago
TDI = Time Domain Integration ?
1
Reply
@wolpumba4099
1 day ago (edited)
Maybe the camera should not be illuminated during readout.
From the datasheet of the sensor (Onsemi): saturation 40300 electrons, read noise 12.5 electrons per pixel @ 18MHz (quite bad). quantum efficiency 62% (if it has micro lenses), frame rate 1 Hz. lateral overflow drain to prevent blooming protects against 800x (factor increases linearly with exposure time) saturation exposure (32e6 electrons per pixel at 4ms exposure time), microlens has +/- 20 degree acceptance angle
i guess it would be good for astrophotography
4
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@txm100
1 day ago (edited)
Babe wake up a new mikeselectricstuff has dropped!
9
Reply
@vincei4252
1 day ago
That looks like a finger-lakes filter wheel, however, for astronomy they'd never use such a large stepper.
1
Reply
@MRooodddvvv
1 day ago
yaaaaay ! more overcomplicated optical stuff !
4
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1 reply
@NoPegs
1 day ago
He lives!
11
Reply
1 reply
Transcript
0:00
so I've stripped all the bits of the
0:01
optical system so basically we've got
0:03
the uh the camera
0:05
itself which is mounted on this uh very
0:09
complex
0:10
adjustment thing which obviously to set
0:13
you the various tilt and uh alignment
0:15
stuff then there's two of these massive
0:18
lenses I've taken one of these apart I
0:20
think there's something like about eight
0:22
or nine Optical elements in here these
0:25
don't seem to do a great deal in terms
0:26
of electr magnification they're obiously
0:28
just about getting the image to where it
0:29
uh where it needs to be just so that
0:33
goes like that then this Optical block I
0:36
originally thought this was made of some
0:37
s crazy heavy material but it's just
0:39
really the sum of all these Optical bits
0:41
are just ridiculously heavy those lenses
0:43
are about 4 kilos each and then there's
0:45
this very heavy very solid um piece that
0:47
goes in the middle and this is so this
0:49
is the filter wheel assembly with a
0:51
hilariously oversized steper
0:53
motor driving this wheel with these very
0:57
large narrow band filters so we've got
1:00
various different shades of uh
1:03
filters there five Al together that
1:06
one's actually just showing up a silver
1:07
that's actually a a red but fairly low
1:10
transmission orangey red blue green
1:15
there's an excess cover on this side so
1:16
the filters can be accessed and changed
1:19
without taking anything else apart even
1:21
this is like ridiculous it's like solid
1:23
aluminium this is just basically a cover
1:25
the actual wavelengths of these are um
1:27
488 525 570 630 and 700 NM not sure what
1:32
the suffix on that perhaps that's the uh
1:34
the width of the spectral line say these
1:37
are very narrow band filters most of
1:39
them are you very little light through
1:41
so it's still very tight narrow band to
1:43
match the um fluoresence of the dies
1:45
they're using in the biochemical process
1:48
and obviously to reject the light that's
1:49
being fired at it from that Illuminator
1:51
box and then there's a there's a second
1:53
one of these lenses then the actual sort
1:55
of samples below that so uh very serious
1:58
amount of very uh chunky heavy Optics
2:01
okay let's take a look at this light
2:02
source made by company Lumen Dynamics
2:04
who are now part of
2:06
excelitas self-contained unit power
2:08
connector USB and this which one of the
2:11
Cable Bundle said was a TTL interface
2:14
USB wasn't used in uh the fluid
2:17
application output here and I think this
2:19
is an input for um light feedback I
2:21
don't if it's regulated or just a measur
2:23
measurement facility and the uh fiber
2:27
assembly
2:29
Square Inlet there and then there's two
2:32
outputs which have uh lens assemblies
2:35
and this small one which goes back into
2:37
that small Port just Loops out of here
2:40
straight back in So on this side we've
2:42
got the electronics which look pretty
2:44
straightforward we've got a bit of power
2:45
supply stuff over here and we've got
2:48
separate drivers for each wavelength now
2:50
interesting this is clearly been very
2:52
specifically made for this application
2:54
you I was half expecting like say some
2:56
generic drivers that could be used for a
2:58
number of different things but actually
3:00
literally specified the exact wavelength
3:02
on the PCB there is provision here for
3:04
385 NM which isn't populated but this is
3:07
clearly been designed very specifically
3:09
so these four drivers look the same but
3:10
then there's two higher power ones for
3:12
575 and
3:14
520 a slightly bigger heat sink on this
3:16
575 section there a p 24 which is
3:20
providing USB interface USB isolator the
3:23
USB interface just presents as a comport
3:26
I did have a quick look but I didn't
3:27
actually get anything sensible um I did
3:29
dump the Pi code out and there's a few
3:31
you a few sort of commands that you
3:32
could see in text but I didn't actually
3:34
manage to get it working properly I
3:36
found some software for related version
3:38
but it didn't seem to want to talk to it
3:39
but um I say that wasn't used for the
3:41
original application it might be quite
3:42
interesting to get try and get the Run
3:44
hours count out of it and the TTL
3:46
interface looks fairly straightforward
3:48
we've got positions for six opto
3:50
isolators but only five five are
3:52
installed so that corresponds with the
3:54
unused thing so I think this hopefully
3:56
should be as simple as just providing a
3:57
ttrl signal for each color to uh enable
4:00
it a big heat sink here which is there I
4:03
think there's like a big S of metal
4:04
plate through the middle of this that
4:05
all the leads are mounted on the other
4:07
side so this is heat sinking it with a
4:09
air flow from a uh just a fan in here
4:13
obviously don't have the air flow
4:14
anywhere near the Optics so conduction
4:17
cool through to this plate that's then
4:18
uh air cooled got some pots which are
4:21
presumably power
4:22
adjustments okay let's take a look at
4:24
the other side which is uh much more
4:27
interesting see we've got some uh very
4:31
uh neatly Twisted cable assemblies there
4:35
a bunch of leads so we've got one here
4:37
475 up here 430 NM 630 575 and 520
4:44
filters and dcro mirrors a quick way to
4:48
see what's white is if we just shine
4:49
some white light through
4:51
here not sure how it is is to see on the
4:54
camera but shining white light we do
4:55
actually get a bit of red a bit of blue
4:57
some yellow here so the obstacle path
5:00
575 it goes sort of here bounces off
5:03
this mirror and goes out the 520 goes
5:07
sort of down here across here and up
5:09
there 630 goes basically straight
5:13
through
5:15
430 goes across there down there along
5:17
there and the 475 goes down here and
5:20
left this is the light sensing thing
5:22
think here there's just a um I think
5:24
there a photo diode or other sensor
5:26
haven't actually taken that off and
5:28
everything's fixed down to this chunk of
5:31
aluminium which acts as the heat
5:32
spreader that then conducts the heat to
5:33
the back side for the heat
5:35
sink and the actual lead packages all
5:38
look fairly similar except for this one
5:41
on the 575 which looks quite a bit more
5:44
substantial big spay
5:46
Terminals and the interface for this
5:48
turned out to be extremely simple it's
5:50
literally a 5V TTL level to enable each
5:54
color doesn't seem to be any tensity
5:56
control but there are some additional
5:58
pins on that connector that weren't used
5:59
in the through time thing so maybe
6:01
there's some extra lines that control
6:02
that I couldn't find any data on this uh
6:05
unit and the um their current product
6:07
range is quite significantly different
6:09
so we've got the uh blue these
6:13
might may well be saturating the camera
6:16
so they might look a bit weird so that's
6:17
the 430
6:18
blue the 575
6:24
yellow uh
6:26
475 light blue
6:29
the uh 520
6:31
green and the uh 630 red now one
6:36
interesting thing I noticed for the
6:39
575 it's actually it's actually using a
6:42
white lead and then filtering it rather
6:44
than using all the other ones are using
6:46
leads which are the fundamental colors
6:47
but uh this is actually doing white and
6:50
it's a combination of this filter and
6:52
the dichroic mirrors that are turning to
6:55
Yellow if we take the filter out and a
6:57
lot of the a lot of the um blue content
7:00
is going this way the red is going
7:02
straight through these two mirrors so
7:05
this is clearly not reflecting much of
7:08
that so we end up with the yellow coming
7:10
out of uh out of there which is a fairly
7:14
light yellow color which you don't
7:16
really see from high intensity leads so
7:19
that's clearly why they've used the
7:20
white to uh do this power consumption of
7:23
the white is pretty high so going up to
7:25
about 2 and 1 half amps on that color
7:27
whereas most of the other colors are
7:28
only drawing half an amp or so at 24
7:30
volts the uh the green is up to about
7:32
1.2 but say this thing is uh much
7:35
brighter and if you actually run all the
7:38
colors at the same time you get a fairly
7:41
reasonable um looking white coming out
7:43
of it and one thing you might just be
7:45
out to notice is there is some sort
7:46
color banding around here that's not
7:49
getting uh everything s completely
7:51
concentric and I think that's where this
7:53
fiber optic thing comes
7:58
in I'll
8:00
get a couple of Fairly accurately shaped
8:04
very sort of uniform color and looking
8:06
at What's um inside here we've basically
8:09
just got this Square Rod so this is
8:12
clearly yeah the lights just bouncing
8:13
off all the all the various sides to um
8:16
get a nice uniform illumination uh this
8:19
back bit looks like it's all potted so
8:21
nothing I really do to get in there I
8:24
think this is fiber so I have come
8:26
across um cables like this which are
8:27
liquid fill but just looking through the
8:30
end of this it's probably a bit hard to
8:31
see it does look like there fiber ends
8:34
going going on there and so there's this
8:36
feedback thing which is just obviously
8:39
compensating for the any light losses
8:41
through here to get an accurate
8:43
representation of uh the light that's
8:45
been launched out of these two
8:47
fibers and you see uh
8:49
these have got this sort of trapezium
8:54
shape light guides again it's like a
8:56
sort of acrylic or glass light guide
9:00
guess projected just to make the right
9:03
rectangular
9:04
shape and look at this Center assembly
9:07
um the light output doesn't uh change
9:10
whether you feed this in or not so it's
9:11
clear not doing any internal Clos Loop
9:14
control obviously there may well be some
9:16
facility for it to do that but it's not
9:17
being used in this
9:19
application and so this output just
9:21
produces a voltage on the uh outle
9:24
connector proportional to the amount of
9:26
light that's present so there's a little
9:28
diffuser in the back there
9:30
and then there's just some kind of uh
9:33
Optical sensor looks like a
9:35
chip looking at the lead it's a very
9:37
small package on the PCB with this lens
9:40
assembly over the top and these look
9:43
like they're actually on a copper
9:44
Metalized PCB for maximum thermal
9:47
performance and yeah it's a very small
9:49
package looks like it's a ceramic
9:51
package and there's a thermister there
9:53
for temperature monitoring this is the
9:56
475 blue one this is the 520 need to
9:59
Green which is uh rather different OB
10:02
it's a much bigger D with lots of bond
10:04
wise but also this looks like it's using
10:05
a phosphor if I shine a blue light at it
10:08
lights up green so this is actually a
10:10
phosphor conversion green lead which
10:12
I've I've come across before they want
10:15
that specific wavelength so they may be
10:17
easier to tune a phosphor than tune the
10:20
um semiconductor material to get the uh
10:23
right right wavelength from the lead
10:24
directly uh red 630 similar size to the
10:28
blue one or does seem to have a uh a
10:31
lens on top of it there is a sort of red
10:33
coloring to
10:35
the die but that doesn't appear to be
10:38
fluorescent as far as I can
10:39
tell and the white one again a little
10:41
bit different sort of much higher
10:43
current
10:46
connectors a makeer name on that
10:48
connector flot light not sure if that's
10:52
the connector or the lead
10:54
itself and obviously with the phosphor
10:56
and I'd imagine that phosphor may well
10:58
be tuned to get the maximum to the uh 5
11:01
cenm and actually this white one looks
11:04
like a St fairly standard product I just
11:06
found it in Mouse made by luminous
11:09
devices in fact actually I think all
11:11
these are based on various luminous
11:13
devices modules and they're you take
11:17
looks like they taking the nearest
11:18
wavelength and then just using these
11:19
filters to clean it up to get a precise
11:22
uh spectral line out of it so quite a
11:25
nice neat and um extreme
11:30
bright light source uh sure I've got any
11:33
particular use for it so I think this
11:35
might end up on
11:36
eBay but uh very pretty to look out and
11:40
without the uh risk of burning your eyes
11:43
out like you do with lasers so I thought
11:45
it would be interesting to try and
11:46
figure out the runtime of this things
11:48
like this we usually keep some sort
11:49
record of runtime cuz leads degrade over
11:51
time I couldn't get any software to work
11:52
through the USB face but then had a
11:54
thought probably going to be writing the
11:55
runtime periodically to the e s prom so
11:58
I just just scope up that and noticed it
12:00
was doing right every 5 minutes so I
12:02
just ran it for a while periodically
12:04
reading the E squ I just held the pick
12:05
in in reset and um put clip over to read
12:07
the square prom and found it was writing
12:10
one location per color every 5 minutes
12:12
so if one color was on it would write
12:14
that location every 5 minutes and just
12:16
increment it by one so after doing a few
12:18
tests with different colors of different
12:19
time periods it looked extremely
12:21
straightforward it's like a four bite
12:22
count for each color looking at the
12:24
original data that was in it all the
12:26
colors apart from Green were reading
12:28
zero and the green was reading four
12:30
indicating a total 20 minutes run time
12:32
ever if it was turned on run for a short
12:34
time then turned off that might not have
12:36
been counted but even so indicates this
12:37
thing wasn't used a great deal the whole
12:40
s process of doing a run can be several
12:42
hours but it'll only be doing probably
12:43
the Imaging at the end of that so you
12:46
wouldn't expect to be running for a long
12:47
time but say a single color for 20
12:50
minutes over its whole lifetime does
12:52
seem a little bit on the low side okay
12:55
let's look at the camera un fortunately
12:57
I managed to not record any sound when I
12:58
did this it's also a couple of months
13:00
ago so there's going to be a few details
13:02
that I've forgotten so I'm just going to
13:04
dub this over the original footage so um
13:07
take the lid off see this massive great
13:10
heat sink so this is a pel cool camera
13:12
we've got this blower fan producing a
13:14
fair amount of air flow through
13:16
it the connector here there's the ccds
13:19
mounted on the board on the
13:24
right this unplugs so we've got a bit of
13:27
power supply stuff on here
13:29
USB interface I think that's the Cyprus
13:32
microcontroller High speeded USB
13:34
interface there's a zyink spon fpga some
13:40
RAM and there's a couple of ATD
13:42
converters can't quite read what those
13:45
those are but anal
13:47
devices um little bit of bodgery around
13:51
here extra decoupling obviously they
13:53
have having some noise issues this is
13:55
around the ram chip quite a lot of extra
13:57
capacitors been added there
13:59
uh there's a couple of amplifiers prior
14:01
to the HD converter buffers or Andor
14:05
amplifiers taking the CCD
14:08
signal um bit more power spy stuff here
14:11
this is probably all to do with
14:12
generating the various CCD bias voltages
14:14
they uh need quite a lot of exotic
14:18
voltages next board down is just a
14:20
shield and an interconnect
14:24
boardly shielding the power supply stuff
14:26
from some the more sensitive an log
14:28
stuff
14:31
and this is the bottom board which is
14:32
just all power supply
14:34
stuff as you can see tons of capacitors
14:37
or Transformer in
14:42
there and this is the CCD which is a uh
14:47
very impressive thing this is a kf50 100
14:50
originally by true sense then codec
14:53
there ON
14:54
Semiconductor it's 50 megapixels uh the
14:58
only price I could find was this one
15:00
5,000 bucks and the architecture you can
15:03
see there actually two separate halves
15:04
which explains the Dual AZ converters
15:06
and two amplifiers it's literally split
15:08
down the middle and duplicated so it's
15:10
outputting two streams in parallel just
15:13
to keep the bandwidth sensible and it's
15:15
got this amazing um diffraction effects
15:18
it's got micro lenses over the pixel so
15:20
there's there's a bit more Optics going
15:22
on than on a normal
15:25
sensor few more bodges on the CCD board
15:28
including this wire which isn't really
15:29
tacked down very well which is a bit uh
15:32
bit of a mess quite a few bits around
15:34
this board where they've uh tacked
15:36
various bits on which is not super
15:38
impressive looks like CCD drivers on the
15:40
left with those 3 ohm um damping
15:43
resistors on the
15:47
output get a few more little bodges
15:50
around here some of
15:52
the and there's this separator the
15:54
silica gel to keep the moisture down but
15:56
there's this separator that actually
15:58
appears to be cut from piece of
15:59
antistatic
16:04
bag and this sort of thermal block on
16:06
top of this stack of three pel Cola
16:12
modules so as with any Stacks they get
16:16
um larger as they go back towards the
16:18
heat sink because each P's got to not
16:20
only take the heat from the previous but
16:21
also the waste heat which is quite
16:27
significant you see a little temperature
16:29
sensor here that copper block which
16:32
makes contact with the back of the
16:37
CCD and this's the back of the
16:40
pelas this then contacts the heat sink
16:44
on the uh rear there a few thermal pads
16:46
as well for some of the other power
16:47
components on this
16:51
PCB okay I've connected this uh camera
16:54
up I found some drivers on the disc that
16:56
seem to work under Windows 7 couldn't
16:58
get to install under Windows 11 though
17:01
um in the absence of any sort of lens or
17:03
being bothered to the proper amount I've
17:04
just put some f over it and put a little
17:06
pin in there to make a pinhole lens and
17:08
software gives a few options I'm not
17:11
entirely sure what all these are there's
17:12
obviously a clock frequency 22 MHz low
17:15
gain and with PFG no idea what that is
17:19
something something game programmable
17:20
Something game perhaps ver exposure
17:23
types I think focus is just like a
17:25
continuous grab until you tell it to
17:27
stop not entirely sure all these options
17:30
are obviously exposure time uh triggers
17:33
there ex external hardware trigger inut
17:35
you just trigger using a um thing on
17:37
screen so the resolution is 8176 by
17:40
6132 and you can actually bin those
17:42
where you combine multiple pixels to get
17:46
increased gain at the expense of lower
17:48
resolution down this is a 10sec exposure
17:51
obviously of the pin hole it's very uh
17:53
intensitive so we just stand still now
17:56
downloading it there's the uh exposure
17:59
so when it's
18:01
um there's a little status thing down
18:03
here so that tells you the um exposure
18:07
[Applause]
18:09
time it's this is just it
18:15
downloading um it is quite I'm seeing
18:18
quite a lot like smearing I think that I
18:20
don't know whether that's just due to
18:21
pixels overloading or something else I
18:24
mean yeah it's not it's not um out of
18:26
the question that there's something not
18:27
totally right about this camera
18:28
certainly was bodge wise on there um I
18:31
don't I'd imagine a camera like this
18:32
it's got a fairly narrow range of
18:34
intensities that it's happy with I'm not
18:36
going to spend a great deal of time on
18:38
this if you're interested in this camera
18:40
maybe for astronomy or something and
18:42
happy to sort of take the risk of it may
18:44
not be uh perfect I'll um I think I'll
18:47
stick this on eBay along with the
18:48
Illuminator I'll put a link down in the
18:50
description to the listing take your
18:52
chances to grab a bargain so for example
18:54
here we see this vertical streaking so
18:56
I'm not sure how normal that is this is
18:58
on fairly bright scene looking out the
19:02
window if I cut the exposure time down
19:04
on that it's now 1 second
19:07
exposure again most of the image
19:09
disappears again this is looks like it's
19:11
possibly over still overloading here go
19:14
that go down to say say quarter a
19:16
second so again I think there might be
19:19
some Auto gain control going on here um
19:21
this is with the PFG option let's try
19:23
turning that off and see what
19:25
happens so I'm not sure this is actually
19:27
more streaking or which just it's
19:29
cranked up the gain all the dis display
19:31
gray scale to show what um you know the
19:33
range of things that it's captured
19:36
there's one of one of 12 things in the
19:38
software there's um you can see of you
19:40
can't seem to read out the temperature
19:42
of the pelta cooler but you can set the
19:44
temperature and if you said it's a
19:46
different temperature you see the power
19:48
consumption jump up running the cooler
19:50
to get the temperature you requested but
19:52
I can't see anything anywhere that tells
19:54
you whether the cool is at the at the
19:56
temperature other than the power
19:57
consumption going down and there's no
19:59
temperature read out
20:03
here and just some yeah this is just
20:05
sort of very basic software I'm sure
20:07
there's like an API for more
20:09
sophisticated
20:10
applications but so if you know anything
20:12
more about these cameras please um stick
20:14
in the
20:15
comments um incidentally when I was
20:18
editing I didn't notice there was a bent
20:19
pin on the um CCD but I did fix that
20:22
before doing these tests and also
20:24
reactivated the um silica gel desicant
20:26
cuz I noticed it was uh I was getting
20:28
bit of condensation on the window but um
20:31
yeah so a couple of uh interesting but
20:34
maybe not particularly uh useful pieces
20:37
of Kit except for someone that's got a
20:38
very specific use so um I'll stick a
20:42
I'll stick these on eBay put a link in
20:44
the description and say hopefully
20:45
someone could actually make some uh good
20:47
use of these things
Example Output:
**Abstract:**
This video presents Part 2 of a teardown focusing on the optical components of a Fluidigm Polaris biotechnology instrument, specifically the multi-wavelength illuminator and the high-resolution CCD camera.
The Lumen Dynamics illuminator unit is examined in detail, revealing its construction using multiple high-power LEDs (430nm, 475nm, 520nm, 575nm, 630nm) combined via dichroic mirrors and filters. A square fiber optic rod is used to homogenize the light. A notable finding is the use of a phosphor-converted white LED filtered to achieve the 575nm output. The unit features simple TTL activation for each color, conduction cooling, and internal homogenization optics. Analysis of its EEPROM suggests extremely low operational runtime.
The camera module teardown showcases a 50 Megapixel ON Semiconductor KAF-50100 CCD sensor with micro-lenses, cooled by a multi-stage Peltier stack. The control electronics include an FPGA and a USB interface. Significant post-manufacturing modifications ("bodges") are observed on the camera's circuit boards. Basic functional testing using vendor software and a pinhole lens confirms image capture but reveals prominent vertical streaking artifacts, the cause of which remains uncertain (potential overload, readout artifact, or fault).
**Exploring the Fluidigm Polaris: A Detailed Look at its High-End Optics and Camera System**
* **0:00 High-End Optics:** The system utilizes heavy, high-quality lenses and mirrors for precise imaging, weighing around 4 kilos each.
* **0:49 Narrow Band Filters:** A filter wheel with five narrow band filters (488, 525, 570, 630, and 700 nm) ensures accurate fluorescence detection and rejection of excitation light.
* **2:01 Customizable Illumination:** The Lumen Dynamics light source offers five individually controllable LED wavelengths (430, 475, 520, 575, 630 nm) with varying power outputs. The 575nm yellow LED is uniquely achieved using a white LED with filtering.
* **3:45 TTL Control:** The light source is controlled via a simple TTL interface, enabling easy on/off switching for each LED color.
* **12:55 Sophisticated Camera:** The system includes a 50-megapixel Kodak KAI-50100 CCD camera with a Peltier cooling system for reduced noise.
* **14:54 High-Speed Data Transfer:** The camera features dual analog-to-digital converters to manage the high data throughput of the 50-megapixel sensor, which is effectively two 25-megapixel sensors operating in parallel.
* **18:11 Possible Issues:** The video creator noted some potential issues with the camera, including image smearing.
* **18:11 Limited Dynamic Range:** The camera's sensor has a limited dynamic range, making it potentially challenging to capture scenes with a wide range of brightness levels.
* **11:45 Low Runtime:** Internal data suggests the system has seen minimal usage, with only 20 minutes of recorded runtime for the green LED.
* **20:38 Availability on eBay:** Both the illuminator and camera are expected to be listed for sale on eBay.
Here is the real transcript. What would be a good group of people to review this topic? Please summarize provide a summary like they would:
Virology Lectures 2026 #6: Synthesis of RNA from RNA
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Viruses with RNA genomes must encode an RNA dependent RNA polymerase because host cells cannot copy viral RNA or make mRNA. In this lecture we discuss the mechanisms of RNA synthesis by RNA dependent RNA polymerases, including priming of RNA synthesis, how mRNA and genomes are produced in infected cells, and the switch from synthesis of mRNAs to genome RNA.
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@JimmyOmba
36 seconds ago
Could I have the PDF of your slides from these virology lecture series? I would like to adapt them into French and present them at scientific gatherings in our medical school ? I'm a medical student from DRC.
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Transcript
0:13
Good afternoon. Welcome back to Viology. So today we will start talking about uh
0:20
events beyond viruses getting into cell. So for the next five lectures, we're
0:27
going to get pretty molecular. And if you don't like that, well,
0:33
sorry, we have to do it. But then after that, we'll talk about disease and stuff, which I think everybody likes. So
0:40
just be patient. So today we're going to talk about RNA viruses making RNA
0:48
synthesis of RNA from RNA and a little bit of history to put this in perspective.
0:54
So back in 1935 a guy named Stanley at the U Rockefeller
1:02
Institute in Princeton he crystallized tobacco mosaic virus. Remember TMV was
1:09
the first virus discovered and he made crystals of it
1:15
and he found a few a year later that these crystals have 5%
1:21
RNA. So he actually got the Nobel Prize for crystallizing
1:27
this virus. But he also said that the RNA was a contaminant and he was pretty
1:33
sure that the protein was the genetic material. I think they should have taken his prize away, but they don't do that.
1:39
They don't do that. That's I think it's one of the worst prizes ever given. But there there are some that are really
1:44
bad, too. Anyway, of course, in 1944, we find out that DNA is genetic material. Avery
1:51
Mloud and ML Avery Mloud and Mccardi working in bacteria. And then the
1:58
Hershey Chase experiment experiment 1952 showing that DNA of a fagee is the
2:04
genetic material. So so much for Wendell Stanley. And then 1953 the structure of DNA was
2:12
solved. That's a propitious year because I was born that year. And I I um blame
2:20
the structure of DNA that year for me being a scientist. There was something about it that seeped into me from that
2:27
year on. uh 1956 the Frankl Conrad experiment that we talked about showing
2:34
RNA as genetic material of a to of tobacco mosaic virus. And so by 1959
2:42
people had identified RNA in many animal viruses. And so um in the 1960s then
2:50
people start to study how this RNA is replicated because there's no precedent
2:55
for that in cells. Okay, cells replicate their DNA. They make RNA like mRNA and
3:01
tRNA and ribosomal RNA, but they don't replicate it. So, this was interesting and people wanted to understand what was
3:08
going on. And so, today I'll give you a couple of examples of viruses making RNA
3:14
from RNA. So, here's our Baltimore scheme and we have mRNA as usual in the middle
3:21
and the viruses in red we're going to talk about today. We'll talk about doublestranded RNA viruses like Rio
3:28
viruses and roto viruses. We'll talk about negative strand RNA viruses
3:34
influenza virus and VSSV and then
3:39
plus strand RNA viruses like polio virus. So you may say what about retroviruses
3:45
they have plus RNA. Well they are unusual that they go through a DNA intermediate. So they have a lecture of
3:52
their own. They're so interesting. So the very first experiments
3:59
uh people did was to understand how RNA viruses could copy their genome. And
4:04
here's an experiment that was done. They take cells in culture. So that's a cell.
4:09
It's not a fried egg. It's a cell. It's a nucleus. And they infect it with polio
4:15
virus. And they let it go for different times after infection.
4:22
uh and then they make an extract of the cell and they add the four triphosphates
4:29
including UTP right which you need for RNA synthesis and one of these is
4:35
radioactively labeled so that you can measure it and you do your incubation and then you measure RNA synthesis. So
4:42
here's the graph of the experiment on the right. So x- axis is hours post
4:48
infection and then on the y- axis is the y- axis on the left is RNA polymerase activity.
4:55
So those are the circles and then on the right is is infectivity pfu per mill and
5:03
those are the squares. So you you infect at time zero of course
5:09
and then you see that the nothing is happening until about two hours. So
5:15
remember that's our eclipse period during which the virus parts are being made. And then if you look at
5:22
infectivity you see from two to three hours is a huge increase and it keeps going up and by four hours the infection
5:30
is over. It's so fast. In four hours it's already peaked and it starts to come down. At the same time if you look
5:36
at RNA sorry that's that's I told you the wrong thing. Let's start again. So between two
5:43
and three hours you have a big production of infectivity. Those are the squares and it keeps going up and it
5:50
really peaks at four to five hours. So it's still still quite rapid. The RNA also goes up and peaks at about four
5:57
hours roughly corresponding with virus production. So that was the first evidence for what we call RNA polymerase
6:05
activity uh in infected cells and subsequently
6:11
the um investigators found for negative strand viruses the RNA polymerase is
6:19
actually in the particle. So you could do an experiment like I just showed you. You could just take purified virus
6:25
particles, crack them open and add the triphosphates and you would get RNA made
6:33
without a cell because the polymerase is in the virus particle. Uh when the se the era of sequencing
6:40
came, we were able to look at the sequences of polymerase proteins. We're
6:46
able to identify them. So there's a very common sequence element glyasp asp which
6:53
if you see in a protein is a good sign that it's a polymerase of some kind when
6:58
we're able to then express recominant proteins and show that they're active
7:04
and finally do molecular structures of the polymerase. So this is where we are today where we can study polymerases and
7:10
we have many many structures. So the uh the different viruses we'll
7:17
talk about today they either do or do not have polymerase in the particle and this is conceptually an important thing
7:23
for you to understand. So first the negative strand RNA genomes
7:29
they have an RNA polymerase in the particle RNA dependent RNA polymerase. A
7:35
negative strand genome has to have the polymerase in the particle because when negative strand RNA gets into the cell,
7:42
it cannot be translated and it can't be copied. So without a polymerase, nothing can happen. So these negative strand
7:49
genomes not only have the polymerase in the particle, of course, the RNA is also coded with protein, typically an N or a
7:55
nucleioapsid protein, and that makes it a nucleioapsid because it's a substructure within the virus. So our
8:03
examples there are VSSV and influenza virus.
8:08
Then our plus strand genomes. These do not have RDRP in the particle.
8:15
We don't need it because when the plus strand comes into cells, it can be translated to make the polymerase. So
8:23
typically not only is there no polymerase, but the RNA is not coded
8:29
with protein. So examples are polio virus, corona v um
8:35
flavy viruses and now there are two exceptions to there being no protein coding the RNA retroviruses
8:43
the RNA is coded with protein so it's a nucleioapsid and the corona viruses are also coded
8:50
with protein but for the most part all the other plus strand RNA viruses there's no protein coding the RNA and
8:56
there's no polymerase and then finally the doublestranded RNA gene genomes the
9:02
RNA is not coded so there's no nucleioapsid structure but there is an
9:08
RNA polymerase in the particle and you may say why is that because it's double stranded it has a plus strand in there
9:16
but it's double stranded so the ribosomes can't get at that plus strand they cannot access it to translate it so
9:23
you need a polymerase in the particle to copy the minus strand and make a messenger RNA so that's the state of RNA
9:31
and RDRP in the virus particle. And here's a picture of nucleioapsids.
9:38
Just to remind you, we now have structures of the protein, the nucleioapsid protein that coats these
9:45
RNAs. Here we have on the upper left. There's a diagram of VSSV. It's an envelope virus with a helical
9:51
nucleioapsid. There's it shown there. And the single end protein is repeated
9:57
many times. and it's bound to the viral RNA. Here on the bottom right in B is
10:03
the structure of the nucleicapsid protein. You can see it's has these two loes and there's a groove in the middle
10:09
and that's where the RNA fits. So all of these protein RNA interactions that's exactly what they look like. And finally
10:16
on the left is a space filling model of many nucleic capsu proteins and uh as
10:23
you can see they form a very nice coil. It's just part of the coil that makes up the particle. And here if you can see in
10:29
this darker blue that's a single nucleioapsided protein. On the upper right is influenza virus which has eight
10:36
RNAs and they are all RNAs complex to protein as you can see on the right here. So nuclear protein. So this is a
10:43
nucleioapsid here on the bottom is the structure of the nucleicapsid protein. Again two loes
10:50
very much like for VSSV and there's a groove in the middle where the RNA fits in very nicely. And then finally on the
10:57
bottom are two space filling views. One from the side and one from the end. So
11:03
you can see it's a tube. These are all nucleic capsid proteins. And there in dark blue is a single nucleic capsuid
11:09
subunit. Just so you can orient yourself. So those are nucleioapsids.
11:14
Now inside the nucleic capsid or inside the particle if there's no nucleicapsid is the RNA of course. And RNA as I
11:22
mentioned before is not just a linear molecule. It's highly structured and these are some of the kinds of
11:28
structures that you would see in RNA. RNA often base pairs with itself to form
11:35
what we call stem loops. So here on the left is a a stem loop structure. So you
11:41
can see the RNA sequence is complementaryary to a sequence just a little bit downstream. So it base pairs
11:48
and it forms what's called a stem loop. And these can be extensive. They can have multiple stem loops. They can be
11:55
interior loops and so forth. Uh and they all have functions. They bind proteins
12:01
and facilitate different parts of the reproduction cycle. They also can form
12:07
what's called a pseudo knot. That's shown in the middle here. This is really interesting. a stem loop
12:15
forms and some of the bases in the loop are able to base pair with with bases
12:22
downstream of the loop. So in in B you can see the interactions drawn with
12:27
dotted lines in in the middle is how they actually uh interact. So the the
12:33
base base pairing in the stem and the base pairing downstream and they actually coil around each other. And
12:40
here on the upper right is the actual structure. So it's called a pseudo knot because it looks like a knot, but it's
12:47
not. It's not an actual string going through it. It looks like it. And um
12:54
many many even these pictures that I give you are wrong. You know, drawing them flat in two dimensions. Um here the
13:01
pseudonut structure is correct. It's three-dimensional. And here at the bottom is the structure of the HIV fivep
13:07
prime end with lots of stem loops. And you can see that its actual structure is really quite different from what we draw
13:13
on the paper. And you'll see these coming up multiple times as we talk in
13:19
this course. So what we have to do today is we have to copy the RNA genome and
13:26
make mRNAs for RNA viruses. Those are the two goals. And there are two rules very simple. The genome has to be copied
13:33
from end to end. Right? So here's an RNA genome from fivep prime to threep prime. You have to copy the whole thing. You
13:39
can't miss a base at the end. It doesn't work. You have to copy everything. And secondly, you have to make mRNAs
13:47
that can be translated by the host cell because you have to make proteins. And for some viruses, if it's a plus strand
13:54
virus, the genome can be the same as the mRNA, but not for all of them as you will see. And of course, for minus
14:01
strand viruses, the genome is never the mRNA. So we have universal rules for RNA
14:07
synthesis. You're going to see the same kinds of rules for DNA synthesis. So here at the top we have a depiction of
14:15
RNA synthesis. So there's our template on the bottom. It has a threep prime and
14:21
a fivep prime end. These are chemically defined ends. And then a polymerase is
14:28
copying the template. uh in this case it's using a primer a short RNA that
14:34
base pairs to the template which the polymerase recognizes and then begins adding bases to the threep prime end of
14:42
the primer based on their complimentarity with the template. And so the chain growth the addition of
14:49
bases is is in the five to three prime direction but the template is read in a
14:56
three to fivep prime direction. Okay? So don't get confused about that. Synthesis is five to threep prime. Copying is
15:02
three to five prime. So uh initiation of RNA happens at a very specific place on
15:10
the template. Sometimes you need a primer and sometimes you don't. If you
15:18
don't need a primer that's called denovo initiation. If you and if you require a primer it's
15:25
not denovo initiation. Other proteins are needed besides the polymerase as you will see and as I said the RNA is
15:33
elongated in a five to three prime direction. Now in most cases the RNA
15:39
synthesis is templated. In other words, the polymerase is copying a template.
15:44
But there are some examples of non-mplated synthesis where for example an RNA will be copying a polymerase will
15:52
be copying an RNA and it bumps into a stem loop and it starts to add bases
15:58
randomly that have nothing to do with the template. So that's non-mplated synthesis. RNA starts with an initiation
16:06
step and there there two kinds of initiation. There's denovo without a
16:11
primer and primer dependent. They're both illustrated here. So at the top we
16:16
simply have a threep prime terminal initiation. The polymerase adds the
16:22
first complimentary NTP to the threep prime base N1 and the second and so
16:28
forth and extends the molecule. So remember when you copy a plus strand RNA
16:34
which is shown here, you're going to make a minus strand. Then two examples of primer dependent
16:40
initiation which we see a lot in RNA viruses. Sometimes there's a primer which is actually a protein. So protein
16:47
primer here in this case there's a protein with a hydroxil and that serves
16:53
to add the first NTP to it. And then there's sometimes capped primers. Caps
16:58
are structures at the fivep prime ends of mRNAs that we will talk about a little bit today and more when we talk
17:05
about translation. But capped primers can also prime RNA synthesis in particular for influenza
17:12
virus as you'll see today. All right. So these are just the initiation step of RNA synthesis. And then as for
17:19
elongation which is shown here there is what's called a two metal mechanism of
17:25
polymerase catalysis and the metal is magnesium.
17:30
Okay. So again we have our polymerase copying a template.
17:36
In this case it's with a primer. The same thing happens with DNA polymerase which is why this is here. But here on
17:43
the right is the template shown chemically. So here's the fivep prime end. Here is the first base. So you have
17:50
the the ribos and a phosphate and another ribos and a phosphate. So that's
17:56
the backbone of the RNA. And then attach attached to each ribos is a base. This
18:02
is of course DNA which is why we have T here but for RNA would be a U. And you
18:08
can see the base pairing between the backbone and the new bases being added.
18:13
So here on the left we're adding the polymerase is adding bases. It's already added a C. It's added an A and it's in
18:21
the process of adding a U which is complimentary to the A. And it looks
18:26
very confusing but let me simplify it for you. So first of all here are two
18:32
magnesiums which are essential for RNA catalysis. They are held in place at the
18:38
active site of the polymerase by two amino acids. In this case they're
18:45
aspartate residues. So there's this one and this one. And those two aspartates
18:50
coordinate the magnesium ions. The magnesium ions facilitate the chemical reactions which remove two
18:59
phosphates and form the the phosphodiester bond. So the the base
19:04
that's come in here it's come in of course as a triphosphate. This would be urodine triphosphate. So we have one two
19:13
three phosphates. uh the magnesiums coordinate this
19:18
triphosphate in the active site and leads to the nucleophilic attack of this
19:23
oxygen on this phosphate which then attacks this the next oxygen and
19:28
liberates two phosphates a pyrophosphate and makes a bond a a phosphodiester bond
19:35
with the base. So that OP bond now makes the the U part of the growing chain and
19:42
you release PP or pyrophosphate two phosphates together which is called
19:48
pyrohosphate. So that's the mechanism of addition. Two metals coordinated by
19:53
amino acids in the active site typically aspartates or something else and then
19:58
that kind of chemical reaction that liberates pyroofhosphate and attaches
20:04
one phosphate to the base. All right, let's see how we understand
20:10
all of this. First question, which is a universal rule about RNA
20:16
directed RNA synthesis? RDRP may initiate denovo or require a
20:22
primer. RNA synthesis initiates randomly on the RNA template. RNA is synthesized
20:28
in a three to fivep prime direction. RNA synthesis is always template directed.
20:35
Okay. How did we do here?
20:41
Most of you got a RDRP may initiate denova or require a primer. It's correct. RNA is not synthesized in a
20:49
three to five prime. It's synthesized in a five to three prime. And it's not always template directed,
20:58
but sometimes it's not. And I will show you
21:03
that slide.
21:09
There is some non-temp templated. I think template is easier if you're
21:14
going to say templated instead of template template. Yeah, it doesn't work.
21:20
There you go. There's some non- templated synthesis and RNA synthesized in a five to three prime direction. You
21:26
copy the template in the three to five prime direction. Okay, let's look at some polymerases. There are four different kinds of polymerases in the
21:33
world in the on Earth that we know of. There is RNA dependent RNA polymerase
21:41
which we're talking about today. There is RNA dependent DNA polymerase that's
21:46
reverse transcriptase. There is DNA dependent DNA polymerase which we have that copies our genome and
21:56
then DNA dependent RNA pulymerase that make that we also have which makes mRNA and tRNA and ribosomal RNAs among
22:03
others. So on the top are just bars indicating
22:08
their lengths and amino acids. You can see the RNA polymerase is the longest 461 amino acids and the others are
22:15
somewhat shorter. And then they have they're alignable. So these four
22:20
polymerases looks like they look like they evolved from a common ancestor many years ago. You can align the sequences
22:28
and in fact there are some very conserved sequences like this red and green and yellow and purple etc.
22:34
that are alignable. Okay. So the the most important for us is this yellow
22:39
which is which has the two magnesium binding sites. So it's part of the catalytic site of the enzyme and you can
22:47
see in here polio polymerase has glyasp asp. So that's a signature uh for an RNA
22:56
polymerase GDD or sometimes allp asin. As you can see, different
23:01
virus polymerase have different signatures, but those two ASPs in the case of the polio virus pulymerase
23:08
coordinate the two magnesiums in the active site that carry out the chemistry I just showed you. Now on the bottom are
23:15
the three-dimensional structures of these four polymerases. So cleno is DNA
23:21
dependent DNA pymerase. T7 RNA polymerase is a DNA dependent RNA
23:27
polymerase. And this is a reverse transcriptase of HIV and the polio virus polymerase. By the way, the T7 RNA
23:34
polymerase, how many of you had mRNA vaccines for COVID? That was made with
23:39
T7 RNA polymerase. How cool is that? It's a fagee polymerase which they can
23:45
do in vitro reactions with. And they added DNA template and it makes you see
23:52
there it said template, a DNA template and it makes RNA. They made 50 kilos of
24:01
mRNA in the first year. We used to do these reactions in my lab. We used to make a
24:07
nanogram at a time. That's all we needed. 50 kilos. It's amazing. Anyway,
24:13
that's T7 RNA polymerase. So, you can see the structures there. And they all
24:18
look like a right hand, okay, which is drawn there where the active site is in
24:24
the palm. The red part is the active site of the enzyme. That's where the catalysis happens. And for some of these
24:30
enzymes, well actually for for the polio polymerase, well, they all have what are called fingers and thumb domains. Fingers,
24:38
thumb, and palm. And for polio polymerase, the fingers and the thumb are touching each other. So they make a
24:45
circle as I think you can see here. Here the fingers domain and the thumb and then they're touching. For the other
24:51
polymerases, they're more open. But that's where the active site is. And in each of these structures, the active
24:58
site is marked by the yellow sequences, right? And the red. So here, for example, is the active site of HIV RT.
25:06
And here for polio polymerase, it's very clear. These two beta strands comprise
25:11
the active site. We're going to take a close look at that in a moment. So having the structure is great because then you can figure out what individual
25:18
parts of the molecule do during catalysis. It's very interesting. So
25:24
here is the polio virus RDRP. Again, a right hand with the thumb and
25:30
fingers domain touching. Here we go at the top here. Here's the active site, the two uh yellow beta strands. And
25:39
there are the two aspartates, the two amino acids, the asp of the
25:45
giasp asp. And that's where the magnesiums are coordinated during
25:51
catalysis. So the active site of course is where RNA is made. Now this of course
25:57
doesn't give you any idea about the geometry of what's happening. So let me try and do that for you.
26:05
These are now space filling models. The previous one was just a ribbon diagram. These are now space filling and we've
26:12
cut away some parts so you can see what's going on. Again a polio RNA dependent RNA pymerase. And what we're
26:19
seeing here is the RNA template uh in green
26:26
going in. We're looking at the left at the top of the molecule. So we're looking top down. The template is going
26:31
in. There's the active site in yellow. Those are the two beta strands.
26:37
The template goes in, makes a right turn and comes out here. And you see it comes out double stranded. So now it has a
26:44
second strand which here is shown in blue or cyan. uh and um so it's it's converted from
26:51
single strand as it passes through the active site. And here we're looking at the front. So
26:58
again the template goes in the top
27:04
goes past the active site which is yellow makes a right turn and now it's coming out of the front of the
27:09
polymerase as a double strand. And now in the back of the polymerase there's a
27:14
very important opening here which is where the triphosphates go in. So the
27:20
triphosphates go in in a different place from the RNA. They go into the active site and they're they're of course
27:26
incorporated. Now what's very interesting is so let's say there's a there's a there's a base at the active
27:32
site part of the RNA. Let's say it's an A. So it's ready to be copied and you have
27:38
to add a U. But the bases are outside the polymerase. So how how does that
27:45
work? It turns out it tries all four of them. And the one that base pairs, boom,
27:51
the catalysis happens. But it really can it does this so quickly that it can go through all four bases. It's like having
27:59
a puzzle and trying to fit things into it. When the right one goes, boom, it fits in and then the catalysis happens.
28:05
So that's how it works. And in fact, sometimes it puts the wrong base in.
28:12
It's very busy and going really fast and making mistakes. It puts the wrong base in. And that's where we get errors
28:17
during catalysis. So, let me show you in in detail how a
28:22
triphosphate fits into the polymerase active site. So, we've zoomed way in.
28:28
Now, we're looking at just the alphaarbon trace with a few side chains.
28:33
So here is one of those active site beta strands. That's the uh the yellow part.
28:40
Here are two ASP molecules, right? D328 and 329.
28:45
Uh and here is a molecule of UTP urodine triphosphate. So here's the ribos there.
28:53
There's the base and then the three phosphates here. And you can see it's
28:59
making non-coovvealent interactions with some amino acids in the active site up here uh down here as well. But what's
29:06
interesting is this particular aspartate not part of the conserved aspartate is
29:12
making a specific uh hydrogen bond with the two prime hydroxil of the UTP.
29:20
So here's UTP drawn here. I flipped it so that it is in the right orientation as to what's in this structure. So RNA
29:27
has two hydroxils at the bottom of the ribos. DNA only has one. This is DNA. This
29:34
would be TTP and so there would be no hydroxil here.
29:39
And that explains why RNA polymerases only use NTPs not deoxynts because that hydrogen
29:48
there can be no hydrogen bond formed with D238. And if that doesn't form, catalysis doesn't happen. So this
29:54
explains why uh our NTPs are preferred and you can see how a triphosphate fits
30:00
uh into the active site. Okay. So let's go through some specific viruses that
30:06
take a bigger picture. We've zoomed really in on the polymerase. Let's back out and look at the picture of how
30:12
things happen. We're going to first talk about plus strand RNA viruses and we'll start with porno viruses. Polio viruses
30:20
are model virus but these are viruses with plus strand RNA genomes
30:27
and the related flavy viruses like West Nile and Zika and Deni they also have
30:33
plus strand genomes they happen to have a fivep prime cap the polio genome doesn't have a cap it has a protein as
30:39
we'll see in a bit those plus strand RNAs are messenger RNAs they can be translated into protein as soon as they
30:46
get into cell so again that's why these viruses don't need to have an RNA polymerase in the particle. Once the
30:53
polymerase is made though, the RNA is copied into a negative strand which is
30:58
then copied to make more plus strands. So those negative strands for these plus
31:03
strand RNA viruses, their sole reason for existing is so they can be copied
31:10
and make more plus strand. They fun have no other function as far as we know uh in the in the infected cell. So let's
31:17
see how this happens in some detail. So here's the overview. Polio virus binds
31:22
the receptor receptor mediated catalysis opens up the particle. So the plus
31:28
strand RNA ends up in the cytoplasm immediately can be engaged by ribosomes
31:36
which make protein. So they'll make a long protein that encompasses the entire
31:41
genome. This genome encodes one open reading frame. makes a very long protein that's then chopped up by proteases that
31:49
are also made by the virus. And among the proteins made are the polymerase and
31:56
accessory proteins. And so they copy this plus strand, the incoming plus
32:01
strand to make double stranded molecules which are plus minus. And those are used to make more plus strands. And the plus
32:09
strands can be used to make more protein or eventually they will go into new virus particles. And this this whole RNA
32:16
synthesis process uh which we'll talk about in a minute happens on membranes
32:22
inside of the cell. When this virus infects the cell, it takes all of the membranes, the internal membranes of the
32:29
cell, the ER and the GG, and it breaks them up. It makes these little double membrane vesicles. And it's on their
32:35
surface that RNA synthesis occurs. So the RNA synthesis is concentrated in a
32:41
specific part of the cell where these vesicles are so that the pulymerase doesn't have to go around finding all
32:47
the components. All right. So that's the overview of what is happening. This is the viral
32:52
genome here. Again a a long RNA 7 and a half KB or so. Threep prime end is
32:58
polyadenilated. Fivep prime end has a protein linked to it and the protein is
33:04
called VPG. That's the primer for RNA synthesis. When this plus strand RNA enters a cell,
33:11
it is immediately translated to make a long precursor which is then chopped up into about a dozen proteins with
33:18
different functions. So here at the threep prime end or all here's the polymerase 3D Paul 3C is a protease. The
33:25
protein at the end of the genome is VPG. It's encoded right there. And then at the fivep prime end or the end terminus
33:32
of the protein we have the capsid protein. So that's the the layout of the
33:37
genome. At the very fivep prime end, there's an untransated region in the genome and
33:44
there's there is a what's called a clover leaf structure. So here's the
33:49
very very fivep prime end. In fact, this is the fivep prime base here. It's it's
33:55
a U. It's linked to the protein by a tyrrosine amino acid to that first
34:02
phosphate. And then the RNA continues and you can see the sequence forms this clover leaf
34:09
structure and then the rest of the genome follows here. And again this protein is the primer for RNA synthesis
34:16
as you'll see now. The RNA actually folds as I said before into various structures. So the there's a clover leaf
34:24
at the fivep prime end. That's very important for RNA. Actually all these structures are important for RNA
34:30
synthesis. In the middle of the genome is an element called the CRE element for
34:36
cis acting RNA element. It is a stem loop structure that can be moved around
34:41
the genome. It doesn't matter where it is as long as you don't disrupt the coding sequence. And then at the threep
34:46
prime end is a pseudon right a pseudon followed by the polya.
34:52
Now these structures are thought to confer specificity for copying to the
34:59
viral RNA because in a polio infected cell the cell RNAs are never copied.
35:06
Only viral RNAs are copied. So there must be something in the viral RNA that attracts the pulmerase and we think it
35:13
is these three signals the clover leaf, the cre and the pseudonop.
35:18
So the first step is to make the primer and that happens on the CRE element. So
35:24
that's the function of the CRE. The polymerase shown here 3CD binds to the
35:29
CRE element. Two two molecules actually bind. Uh and then a molecule of VPG
35:36
comes in and the polymerase adds two U's to it. This top loop of this CRE element
35:42
is all A's. So it's basically used as a template to add U's to the VPG. So now
35:48
we have VPG UU which is the primer for RNA synthesis.
35:54
And here's how it works. At the top we have the viral RNA, the plus strand that's come into the cell.
36:01
It's actually attached to membranes via viral protein. But here you see our
36:07
three our fivep prime clover leaf cre element pseudon at the threep prime end.
36:13
First thing that happens is of course VPG is uradilated on the cre which I've
36:18
already shown you and then the VPG is used by the polymerase to start copying
36:24
at the threep prime end which is polyadenilated. So those two 's line up nicely uh with the threep prime poly.
36:31
The RNA is actually circularized during the initiation step. There is a cell protein called poly aa binding protein
36:39
that binds the RNA binds the poly specifically. It also binds the clover leaf. So basically loops the RNA around
36:46
itself and somehow that allows the polymerase to start initiating with VPG
36:52
at the threep prime end. Then the next step we're we're seeing the priming of
36:57
of the VPG UU on the threep prime end and then eventually extension and
37:03
formation of a doublestranded molecule. So the polymerase will run through this whole plus strand and make a minus
37:08
strand which remains bound to the plus strand as a double stranded form and then the minus strand is copied in a
37:15
similar fashion to make more plus strands. So the keys here are protein priming and a loop formation of the RNA
37:23
template. So the next question is what's which is part of the polio virus
37:29
replication strategy the production of subg genomic mRNAs
37:34
denovo without primer initiation of RNA synthesis circularization of template
37:40
for initiation of RNA synthesis or all of the above. So subgenomic
37:46
simply means shorter than the genome. Okay, we seem to be stuck at 19. So
37:52
let's see how we did. Most of you, well half of you, less than
37:57
half got C. So this was a tough one. Okay. Yeah, a lot of you picked all of
38:03
the above. So let's go through it. The production of subg genomic RNAs. There's
38:09
no subg genomic RNA. Every RNA is just a copy of the original one. uh that that
38:15
comes in the cell denovo initiation. The VPG is the primer. So this is a primer dependent
38:21
polymerase. VPG with two U's on it is the primer. Circularization is needed.
38:27
Many of you got that. That's correct. And so it's not all of the above. So let's have a look at this to
38:33
reinforce what you should have gotten from it. So
38:40
the primer for synthesis is VPG UU.
38:46
The polymerase which is shown here is copying the whole plus strand to make a
38:51
minus strand. So there's no subg genomic RNA. What was the other one?
38:57
Primers needed. No subgenomic RNA. That's it. Okay.
39:03
Now let's look at some flav viruses. I'm sorry. Let's look at flavies are
39:08
very similar to polio. Let's look at some alpha viruses that have a subgenomic RNA here in their synthesis.
39:14
They have a plus strand genome. They copy it to make a minus strand and from the minus strand they make a subgenomic
39:20
mRNA which is shorter than the full genome. That's what subgenomic means.
39:26
So here's the replication the reproduction cycle in the cell. The virus attaches the receptor endoccytos.
39:33
The RNA gets into the cytool where it's translated into proteins. Some of the
39:39
proteins induce the formation of these vesicles on which RNA is made. So you go from plus to minus to plus
39:48
including the synthesis of a subgenomic RNA. So let's take a look at how that happens. So here's a viral genome coming
39:55
into a cell. This genome is capped and polyadenilated.
40:02
It's initially translated but only about half of it is translated into protein.
40:09
These proteins at the top P123 P1234 make up the RNA polymerase and the
40:15
proteins needed for RNA synthesis. So they will copy the input RNA to make a
40:21
negative strand. Once the negative strand is made, the polymerase then makes a subgenomic RNA
40:30
and that's used to make the structural proteins, the capsid and the glyoproteins.
40:36
So it's different from polio virus in that it has this subgenomic RNA.
40:43
Now I you're going to ask why why does this happen? Why doesn't it do the same thing as polio? And I don't we don't
40:52
know why you know why questions in biology are very hard to answer but it works. So
40:59
evolutionarily at some point there was a branch viruses that do or do not make a
41:04
subgenomic RNA. They both are able to compete. They both work so they both exist. That's the best answer we can
41:11
give you. Now corona viruses I include because obvious reasons
41:17
because they SARS Kovv2 has been a very important virus and will continue to be
41:23
these viruses again enveloped bind receptors the RNA gets put into the
41:28
cytool and again similar to the flaves only about half of the genome is
41:33
translated to make the proteins needed for RNA synthesis. So you induce the
41:39
formation of membrane vesicles. You can do genome replication through a minus strand. And these viruses also make
41:45
subgenomic mRNAs. So the the viral genome is about 30,000
41:50
bases long. It's very long. And the first half or so is translated. Here's
41:56
the whole genome here in B. The left half is translated to these replication
42:01
proteins. But there are other proteins encoded to the right. And those are all
42:07
made from subgenomic mRNAs. And there are a lot of them. One, two,
42:13
three, four, five, six, seven, eight of them in this corona virus. And here's our spike protein here.
42:21
Nucleio capsid protein down at the bottom etc. Now you can see these range in size from very big to the smallest
42:30
only encodes the N protein. The second one encodes M andN but only M is
42:36
translated. The first protein, the first open reading frame is the only one translated E protein S A 4 five
42:48
sorry spike H E and 2A. So the first open reading frame is the only one
42:54
translated in these messenger RNAs. So lots of subgenomic mRNAs. Now these
43:00
viruses do something that no other v RNA virus does and that is they make these
43:07
subg genomic RNA mRNAs in a very weird way. So here's the plus stranded genome
43:13
at the top. Here's the RNA polymerase which is starting to copy it making a negative
43:19
strand. When this polymerase gets to close to this first sequence, this blue
43:25
sequence, it's a termination sequence. The RNA actually loops back on itself.
43:31
The polymerase then jumps to the fivep prime end of that RNA and then sticks
43:37
this gray portion onto that mRNA. This is actually a negative strand. So it's not an mRNA yet. Okay. So by this
43:45
looping around the polymerase adds what's called a leader sequence to each mRNA. So then you end up with a negative
43:53
strand copy of the mRNA with this leader. Those are then copied into plus strands which are the messenger RNAs.
44:00
And the reason we can make seven or eight mRNAs is because the polymerase terminates at these different
44:06
termination sequences here. And each of them have a common fivep prime leader.
44:12
Now the key here is that the polymerase is actually going from copying the the
44:18
threep prime end of the minus the plus strand to the fivep prime end of the
44:23
plus strand. So it's changing templates that's recombination basically. So these
44:30
viruses are very good at recombining and that's why they have such pandemic
44:35
potential because in nature different corona viruses recombine at very high
44:41
frequencies and SARS KV2 is a recombinant of multiple different corona
44:46
viruses that circulated in bats. So this recombination is really important for diversity.
44:53
All right. Now negative strand viruses we have two flavors here. We have viruses with a single RNA uniocular
45:02
and viruses with segmented genomes. And so the genome here is in this olive
45:08
color in the middle. And so that genome goes into the cell with a polymerase
45:15
that catalyzes the synthesis of both mRNAs and a plus strand complement to make
45:22
more minus strand genome RNAs. So let's see how that happens for VSSV and flu.
45:29
VSV again entering the cell. The RNA genome coded with protein gets into the
45:35
cytool. The polymerase makes mRNAs, subg genomic mRNAs which give rise to
45:41
proteins. The polymerase will also copy the negative strand into a plus strand
45:46
and then make more minus strands and of course those can go on to make new virus
45:52
particles. Now something new here that we didn't encounter with the plus strand viruses. when the genome is not
45:58
messenger RNA, there has to be a switch at some point from making mRNA to making genomes. So,
46:07
as soon as the RNA gets into a cell, it makes mRNAs because it needs to make proteins to get going. But once it has
46:14
enough proteins to assemble virus particles, then it needs to make
46:20
something to put in it, a negative strand genome. So there has to be a switch from mRNA synthesis to genome.
46:26
We're going to see how that happens for these various viruses. So here's VSSV, a bulletshaped particle as we've seen
46:33
before with an negative strand genome wrapped up in a nucleic capsuid protein has an RNA polymerase in the particle.
46:40
Here's the genome RNA. It's a long negative strand RNA and it gives rise to
46:45
multiple subgenomic mRNAs. As you can see here, they're all capped and polyentilated and they encode the
46:53
various viral proteins. So here is what's what happens when the
46:59
RNA gets into a cell. So the the negative strand is shown here. That's the strand that's in the genome that's
47:06
in the particle. It's coated with nucleioapsid. Those are those circles on it. The polymerase that's brought in
47:13
with the particle initiates at the threep prime end and starts making mRNAs.
47:19
And then at some point when proteins enough proteins have been made, the
47:25
polymerase switches from making mRNA to making a fulllength plus strand. Right?
47:31
Because you can't make a new virus with mRNAs. You need a full length minus
47:37
strand. So you make a plus strand and then finally a minus strand. The the
47:43
protein that causes the switch from mRNA synthesis to fulllength plus strands is
47:50
the N protein. When N proteins are low in the in the beginning of infection,
47:55
the polymerase makes RNA mRNA. When n p when n end protein levels rise, then
48:03
they begin to coat the product and the polymerase makes a fulllength plus
48:08
strand. So let's take a look at that in some more detail. So again, here's the
48:14
negative strand RNA. There's our polymerase binding to the threep prime end. It synthesizes the mRNA for the
48:21
first gene. It stops at an intergenic region, then stops again and makes the
48:27
next gene. and so on uh down the line
48:33
at the intergenic region polyadenilation occurs. So here's a zoom in of the
48:39
intergenic region. There's our negative strand genome. The pymerase has just finished making one mRNA and it
48:46
encounters this stretch of U's in the intergenic sequence and it begins to
48:53
slip and add A's. So adds about 200 A's
48:59
because it's just slipping on this U sequence and then finally it terminates
49:04
releases this now as a polyadenilated RNA and it can begin the next one. So it's an unusual mechanism of
49:10
polyentilation where the polymerase is slipping on this U sequence and it's basically non-mplated for the most part.
49:17
There only seven U's there yet the enzyme can add 200 A's or so.
49:23
Okay. So now influenza virus slightly different because the RNA genome is segmented but some of the same
49:30
principles. Um again the genome is not mRNA so there has to be a switch from making mRNA to making genome synthesis.
49:38
This virus releases its RNA in the cytool as we saw in the animations last
49:43
time. And those RNAs go in the nucleus. So unlike all of the RNA virus we've
49:49
talked about so far, this one goes in the nucleus where they are copied to
49:54
form messenger RNAs that go out into the cytool and make various proteins and
50:00
then at some point they're copied to fake make fulllength plus strands that are copied to minus strands that can
50:06
then be incorporated into virus particles. So there has to be a switch from mRNA to genome synthesis. And
50:13
again, it's controlled by the end protein. So the viral genome is segmented. It has eight negative strands
50:21
which each encode one or two mRNAs that encode a couple of different proteins.
50:27
And again, they're coded with protein in the genome in the virus particle. So they are a nucleioapsid.
50:36
Here's the replication scheme. Here's our viral RNA coming into the cell. It's coated with nucleioapsid.
50:43
The polymerase needs to make an mRNA from this. So this is one of the eight segments.
50:49
The polymerase uses a primer. It is a piece of host mRNA. So host mRNAs all
50:56
have a cap at the fivep prime end. The viral polymerase includes an enzyme that
51:02
cuts host mRNAs and produces these primers. They're about 11 to 12 bases
51:08
and they serve as the primer for mRNA synthesis. So every influenza virus mRNA
51:14
in fact has a little bit of host mRNA sequence at its fivep prime end but it doesn't code for anything. Again when
51:21
the nucleapsured protein levels get high the enzyme switches from making mRNAs to
51:28
fulllength plus strands which can then be made into minus strands. Now, if you
51:33
notice here, the mRNA is different from the genome. First of all, it has this
51:39
cap at the fivep prime end, and it's also about 20 bases short at the threep
51:44
prime end. So, you could never use that mRNA as a template to make minus strands. That's why you have to make a
51:50
fulllength plus strand intermediate.
51:55
And how that works is very interesting. There's so as I said the polymerase of the particle has an enzyme that cleaves
52:04
host mRNAs to make the primer. So here's the cap structure and then there 11 to
52:09
13 bases in the primer and the polymerase will match that up with the threep prime end of the minus strand and
52:16
begin to elongate it. So this endonuclease that carries that out that's a viral protein. There is now a
52:24
new flu antiviral that inhibits that. So many of you may have had Tammy flu in
52:31
the past when you had confirmed influenza. There's a new one called zofluza which
52:37
is an inhibitor of the endonucleus. It's a different target from Tammy flu and in
52:43
fact it's only a one pill deal whereas Tammy flu you have to take for five
52:49
days. So it's it's actually a better antiviral. Now polyadentilation happens in this
52:56
way. It's quite interesting. So the negative strand here's the here's the RNA polymerase of influenza virus. It's
53:02
got multiple subunits PA, PB1 and PB2.
53:07
The viral RNA template is bound at the fivep prime end to the polymerase.
53:14
And the RNA actually is drawn through the active site. This is the active site here.
53:22
And in the active site, of course, the the mRNA is made. So imagine it starts
53:27
with the threep prime end here and the RNA is pulled through this active site. So this is a case where the enzyme
53:33
doesn't actually move along the template, but rather it just stays in one place and the template is pulled
53:39
through and the mRNA is made. The problem is at the very end
53:45
you can't pull anymore. You've run out of flex here. But it turns out there's a stretch of
53:51
U's there. So the polymerase just starts cranking out A's and that's your poly A
53:58
sequence just very similar to VSSV except here the steeric hindrance in
54:05
preventing the polymerase from copying to the end because the five prime end is attached. So it can't pull through anymore yet. That is the mechanism of
54:12
polyadentilation. Okay, next question. How are influenza
54:17
virus and VSSV RNA synthesis similar? A.
54:22
The switch from mRNA to genome RNA synthesis is controlled by an RNA
54:29
binding protein. B. Polyentilation occurs at a short
54:34
stretch of U residues. C. Viral mRNAs are shorter than negative strand genome
54:41
RNA. D. All of the above. Okay, let's see how we did.
54:48
So the answer is all of the above. 60% of you got that.
54:57
You can change your answer once you see it right. A is going down and down
55:04
to switch from mRNA to genome is controlled by an RNA binding protein. That's right. But it's not the only
55:10
thing that's right. Poly dentenilation occurs at a short stretch of use. Right. the pulling the template through it
55:16
can't go anymore. There's a stretch of views there. So, it's polyanilated. Viral RNAs are shorter than
55:23
the genome RNAs. I'll show you show you that here's our viral genome RNA and the
55:29
mRNA is shorter by about 20 bases at the threep prime end. Why is that? Because
55:35
you can't pull the template through anymore here. That's about 20 bases from
55:41
the end of the mRNA to the fivep prime end there. It just cannot physically pull it through. That's why it's
55:47
shorter, but it's polyentilated and it's a messenger RNA. It's okay.
55:52
When it's time to make plus strands, the enzyme is fully able to go all the way
55:57
to the end. And finally, Rio virus is double stranded RNA, which is unusual because
56:04
the RNA has a plus and a minus strand, but the plus strand can't be translated
56:09
by ribosomes. It's inaccessible. So these viruses carry an RNA polymerase
56:15
into the particle. And so the doublestranded RNA is copied to form an
56:20
mRNA that can be translated to protein or the mRNA can be copied again to make
56:28
a genome. In this case, the mRNA is a complete copy of the N minus
56:34
strand. So there's no problem in using the mRNA either for translation or for
56:39
replication. Here's the the Rio virus genome. Remember there are three kinds of
56:46
particles. There's a double shell viron. There's the ISVP where the outer shell is removed partially and then the core
56:53
where only the inner shell remains. And this is the one that pops out of the endoome. We have multiple doublestranded
57:00
RNA segments, each of which encode an mRNA that makes one or more proteins.
57:08
So here's how it happens. The virus, remember, goes into the endocyic pathway. The outer shell is removed and
57:16
the core penetrates into the cytoplasm because it's very hydrophobic.
57:22
those RNAs inside those doublestranded RNAs never leave. So this is an example
57:27
of a virus that never releases its genome rather the polymerase begins to
57:32
copy the doublestranded RNAs and the messenger RNAs come out of these turrets at each five-fold end. You can see them
57:39
coming out there and those mRNAs of course can be made into protein.
57:46
Eventually some of those mRNAs are packaged into new virus particles. So that's just singlestranded mRNA there
57:54
and in that particle is the polymerase also. So it makes it double stranded and the virus particle can be completed and
58:01
released from the cell. So all the mRNA syn all the RNA synthesis of this virus
58:08
happens within the capsit. Nothing is happening uh in the cytoplasm.
58:15
And why might that be? Probably to evade nucleic acid sensors which would lead to
58:22
uh inhibition of virus replication. And we'll talk about that uh later.
58:28
So here's a picture of Riovirus actually this is a rotovirus but same idea
58:34
releasing mRNA from the particles. So they purified particles and they incubated it so that the polymerase
58:41
would make mRNA. And you can see the mRNA is coming out of each five-fold axis of symmetry. The red is the cap on
58:48
the fivep prime end of the RNA. And and this and these particles the uh the
58:54
polymerase there's one polymerase molecule right underneath each five-fold
59:00
axis which is where these turrets are. I showed you in the previous slide these
59:05
turrets which are exposed. The mRNA eventually comes out of those. There's a polymerase molecule at the very bottom
59:12
of each turret. So why do you think no doubleranded
59:20
virus RNA virus has ever been found with more than 12 segments?
59:28
Does 12 ring a bell to you? Yes, there are 12 five-fold axes in the aosahedron.
59:34
So there's one polymerase and one RNA segment attached on the inside to each
59:40
five-fold axis. So that model for synthesis is probably right.
59:45
All right, last thing we talk about is making mistakes. You know, making mistakes is the way to move forward.
59:52
It sounds weird, but it is. Viruses make mistakes and that's how they evolve.
59:58
In RNA viruses they make errors in misinccorporation of nucleotides. RNA
1:00:04
viruses except for corona viruses do not have error correction machinery. So all
1:00:09
polymerases make mistakes but DNA polymerases have error correction. RNA polymerases do not. So they make a
1:00:17
mistake in every thousand to 10,000 bases polymerized. So in an RNA of 10 KB
1:00:25
in a virus, if you have a mutation frequency of 1 in 10,000, you get one
1:00:30
mutation per genome. So and remember there are many genome cycles in an infected cell. So you make lots and lots
1:00:38
of mutations. Some of them kill infectivity, some of them are neutral, but some of
1:00:44
them may be advantageous in some situations. All viruses do this, but RNA
1:00:50
viruses do it more than others. Now the neato virales which is an order
1:00:55
including the corona viruses do have an error correcting protein. It's called exo and it's an exonucleus. It can
1:01:02
detect when the wrong base is put into a growing chain. It will cut it out and
1:01:07
then the polymerase will fix it. And so if you take the exo gene out of a corona
1:01:14
virus you get a 15 to 20fold increase in the mutation rate. So it's very important for keeping the mutation rate
1:01:19
down. And we think this is why the coronavirus genomes are very long. We've seen them up to 40 KB probably because
1:01:27
of the error correction uh machinery. Now there are we know a little bit about
1:01:34
what aspects of the polymerase control the fidelity of the enzyme. For polio
1:01:39
polymerase it's all happening at the active site of the enzyme. Remember the
1:01:45
NTP the NTPs are swapped in one by one until one is found that fits. If it's
1:01:52
the wrong NTP, it will not will not interact with the two espartates here. And so it's it's
1:02:01
rejected. But when the right triphosphate comes in, it's correctly base paired. you will then have a
1:02:08
reorientation of the triphosphate in the pocket and that allows it to be incorporated into the growing chain. So
1:02:14
only the right base pairing will lead to polymerization although as I said one in
1:02:20
10,000 times it makes a mistake. This is happening really really quickly. So a
1:02:26
lot of room for error. So amino acid changes have been introduced into the
1:02:31
RNA polymerase of polio virus which control its fidelity its ability to make
1:02:37
mistakes. So for example a single amino acid change in the polymerase at this position 64 from glycine to serene and
1:02:45
there's there it is right there. It makes the polymerase make fewer
1:02:52
mistakes. it makes a more faithful polymerase and what it does it slows the
1:02:57
confirmational change that occurs when the right NTP is pairing it reduces the
1:03:04
elongation rate and that seems to be a factor in allowing the wrong base to be
1:03:09
present. So this is very interesting because look the this 64 amino acid is
1:03:14
very far away from the active site yet apparently it can make changes in the active site that regulate the uh the
1:03:22
fidelity. Now these enzymes are very interesting. You can make them in the lab but they
1:03:27
don't exist in nature. They cannot survive. They have low they have low fidel um they have low fitness and so
1:03:36
they're never selected for in nature. You never select for enzymes that make fewer mistakes because mistakes making
1:03:43
mistakes is part of evolution. RNA viruses also recombine
1:03:49
and that happens as I've already mentioned when a polymerase is copying one RNA it can switch to another and
1:03:57
therefore you make a hybrid sequence. This open this happens very frequently
1:04:02
for example in uh polio virus and related virus infected cells up to 20%
1:04:07
of RNA molecules recombine in a polio virus infection. So for example in the
1:04:14
old days we used to take an oral polio vaccine it would replicate in our intestines. Turns out our intestines are
1:04:21
full of related viruses that are not pathogenic and they recombine with the
1:04:26
vaccine and the consequence is that the vaccine can reacquire the ability to
1:04:32
cause paralysis. We'll talk about that later. So recombination is huge in shaping the RNA virus world and we saw
1:04:40
we saw that for corona viruses as well. Recombination is also controlled by the
1:04:46
polymerase. There are four amino acids that have been identified uh that control recombination frequency. You can
1:04:53
see here they're in the thumb domain and this is where the RNA comes out of the
1:04:59
polymerase and they're thought to slow down the polymerization
1:05:04
and make the polymerase able to distinguish between homologous and
1:05:10
non-homologous templates and they reduce the amount of recombination. Now this is very interesting. We're
1:05:16
going to see in ne in a few weeks um people modified the polio vaccine to
1:05:24
contain these mutations that these amino acid changes that reduce recombination
1:05:29
and reduce mutations because they wanted to stabilize the polio vaccine. But what
1:05:36
happens is that virus goes into the gut of recipients and all the mutations are
1:05:41
recombined out and the virus is now as fit as it was before. The Gates
1:05:48
Foundation paid scientists $4 billion dollar to do this to make a more stable
1:05:55
polio vaccine. And as soon as it goes in your gut, all the mutations are removed.
1:06:01
We'll talk about that later. You can't fool nature. Next time we're going to switch to DNA
1:06:08
viruses. And the first thing we're going to talk about is making mRNA.