Posts Tagged ‘polymerase’
Covid 19: How the SARS-CoV-2 virion does its thing
Canto: We’ve been lapping up the excellent Medcram series of videos on the pandemic, and we’re now at episode 32 I think, from March 6, and a week’s a long time in Covid-19-world.
Jacinta: Yes and back then the largest number of confirmed cases outside of China was in South Korea, and that, I now understand, was largely because of the massive testing they’d engaged in – so elsewhere the infection was being under-reported, or barely known about.
Canto: And today, April 23, South Korea has dropped down to 27th on the list of reported cases. Interesting to note that by March 6 South Korea had tested some 140,000 people, almost 100 times more than the USA had done. As we know, the CDC had stuffed up by producing a flawed testing kit, which resulted in crucial delays.
Jacinta: And weren’t the South Korean tests more effective? They used a different type of test didn’t they?
Canto: According to a Bloomberg article referred to in the video, South Korea’s tests had a 95% sensitivity rate, much higher than those of the USA at the time. But neither the article nor the video went into detail about the type of test.
Jacinta: So I think the standard type of test used is called PCR, or RT-PCR, which means reverse transcriptase polymerase chain reaction, but I don’t really know what that means or how the tests work.
Canto: We’ll look at how the tests work later. Let’s use this video 32 to help us understand how this virus gets into a host cell and replicates.
Jacinta: Ok, so we have a cell with its nucleus, and its DNA in there, and outside the nucleus is the cell’s cytoplasm containing organelles such as ribosomes, mitochondria, lysosomes, microtubules and the like. The DNA is transcribed into single-stranded precursor messenger RNA. The RNA is then transported into the cytoplasm, where it’s modified, giving it a ‘five prime cap and a poly-A tail’. So one end has its nucleotide altered by the enzyme guanyl transferase. It has to be a guanine nucleotide connected to the mRNA with a particular triphosphate linkage. The poly-A tail is a string of adenine bases. These modifications form what’s called post-transcriptional RNA processing. Then the ribosome, about which we’ve learned so much from Venki Ramakrishnan, reads the mRNA from the five-prime end to the three-prime end. That’s in the ‘positive’ direction. It reads the nucleotides three at a time and comes up with a code (here it gets a bit vague), so that when three particular nucleotides line up, ‘a specific amino acid has to be placed on there’. And transfer RNA is involved here. So a by-product of this process is a protein (consisting of amino acids), made by the ribosome. That’s translation, not so clearly explained. Anyway, proteins are the central building blocks of our bodies, without which not.
Canto: Okay, sufficient unto the day. And remember, this transcription/translation process is known as ‘the central dogma of molecular biology’, in case you’re tested. Now we’ll turn to the virion. So the cell membrane that the virus needs to penetrate is a lipid bilayer. That bilayer is hydrophilic on the outside (that’s facing out from the cell and into the cell) and lipophilic on the inside. The coronovirus has the same lipid bilayer, with embedded proteins, notably the s-proteins or spike proteins which we know are used to attach to host cells. There are other structural proteins such as m-proteins (membrane proteins) and e-proteins (envelope proteins). Inside is the large RNA genome, protected by n-proteins (nucleocapsid proteins). Presumably there are other proteins too. Now, note that this is one virion, which is the built structure housing the virus (what enables it to survive for however long outside of a host), but also including the virus itself, which is essentially the genome. For the virus to replicate and spread, all those structural proteins have to be reproduced too.
Jacinta: The s-protein just happens to fit, like a key in a lock, a receptor protein in the human host cell membrane called the ACE-2 receptor. These ACE-2 receptors, full name angiotensin-converting enzymes, are found in our lungs, and elsewhere, such as the heart, the kidneys and the intestines. Once this connection is made, the viral RNA is released into the cytosol. And as it happens, this viral RNA also has a 5 prime cap and a poly-A tail just like the host’s mRNA. It isn’t clear from the video whether this is because it gets modified within the cytoplasm or it’s already ‘primed’ so to speak. Anyway, the cell’s ribosomes start to act on this rogue RNA as it would on its own mRNA. Meanwhile the structural proteins from the viral membrane are incorporated into the host membrane, possibly earmarking it for destruction.
Canto: The ribosome makes a protein from the viral RNA, called RNA-dependent RNA polymerase (RdRP), or an RNA replicase. The protein somehow makes another complementary strand of RNA, running in the opposite direction, from which the ribosome makes more protein, which makes more RNA and so forth. This RNA also codes for the structural proteins of the virion (because the RdRP somehow forms shorter strands of RNA, called sub-genomic RNAs, specific to the making of those proteins by the hijacked ribosomes), so enabling the spread of the virus.
Jacinta: The key, the video tells me, is in the name polymerase. That’s an enzyme that puts nucleotides together in long chains. Also, many ribosomes – there are thousands in our cells – are connected to the cell membrane and can help create new virions that can leave the cell in much the opposite way they entered, being packaged and then budded off. Through this hijacking process, one virion can come in, and any number of them can go out, and generally from the lung region. They’re naturally attacked by the immune system causing inflammation, possibly pneumonia and respiratory failure.
Canto: Yes and thanks to Dr Roger Seheult for all this, we hope we’re not misreading his work. He goes on to talk about the possibility of inhibiting this nasty polymerase, RdRP. We might talk about this, or not, in the next post.
References
Coronavirus update 32, with Dr Seheult – series of videos
The gene machine, by Venki Ramakrishnan
a DNA dialogue 6: Okazaki fragments, as promised
Canto: Okay, so first off, why are Okazaki fragments so called?
Jacinta: Well as anyone would guess, they’re named after someone Japanese, in this case two, the husband and wife team Reiji and Tsuneko Okazaki, who discovered these short, discontinuously synthesised stretches of DNA nucleotides in the 1960s.
Canto: Yes their story is intriguing – tragic but also inspiring. Reiji, the husband, was born in Hiroshima and died in 1975 from leukaemia, related to the 1945 A-bomb. He was only 44. Tsuneko Okazaki continued their research and went on to make many other contributions to genetics and molecular biology, as a professor, teacher, mentor and director of scientific institutes. Her achievements would surely make her a Nobel candidate, and she’s still alive, so maybe…
Jacinta: Now the key to Okazaki fragments is this lagging strand. Its directionality means that the DNA primase, followed by the DNA polymerase, must work ‘backwards’, away from the replication fork, to add nucleotides. This means that that they have to have periodic breaks – but I’m not sure exactly why – in creating this lagging strand. So the entire replication process is described as semi-discontinuous because of this fundamental difference between the continuously created leading strand and the stop-start ‘fragmentary’ (at least briefly) lagging strand.
Canto: But we need to know why this ‘backward’ movement has to be stop-start, and I’d also like to know more about this primase and polymerase, thank you.
Jacinta: Well the Okazakis and their team discovered this semi-discontinuous replication process in studying the replication of good old Escherichia coli, the go-to research bacterium, and it was a surprise at the time. Now, I’m looking at the explanation for this necessarily discontinuous process in Wikipedia, and I confess I don’t really understand it, but I’ll give it a go. Apparently the Okazakis ‘suggested that there is no found mechanism that showed continuous replication in the 3′ to 5′ direction, only 5′ to 3′ using DNA polymerase, a replication enzyme’, to quote from Wikipedia. So they were rather cleverly hypothesising that there must be another mechanism for the 3′ to 5′ lagging strand, which must be discontinuous.
Canto: And another way of saying that, is that the process must be fragmentary. And they used experiments to test this hypothesis?
Jacinta: Correct, and I won’t go into the process of testing, as if I could. It involved pulse-labelling. Don’t ask, but it has something to do with radioactivity. Anyway, the test was successful, and was supported by the discovery shortly afterwards of polynucleotide ligase, the enzyme that stitches these fragments together. Now, you want to know more about primase, polymerase, and now ligase no doubt. So here’s a bit of the low-down. DNA primase is, to confuse you, an RNA polymerase, which synthesises RNA from a DNA template. It’s a catalyst in the synthesis of a short RNA segment, known as a primer. It’s extremely important in DNA replication, because no polymerase (and you know how polymerase keeps getting associated with primase) can make anything happen without an RNA (or DNA) primer.
Canto: But why? This is getting so complicated.
Jacinta: I assure you, we’ve barely scratched the surface….
Canto: Well, Socrates was right – there’s an essential wisdom in being aware of how ignorant you are. We’ll battle on in our small way.
a DNA dialogue 5: a first look at DNA replication
Jacinta: So let’s scratch some more of the surface of the subject of DNA and genetics. A useful datum to remember, the human genome consists of more than 3 billion DNA bases. We were talking last time about pyrimidines and purines, and base pairs. Let’s talk now about how DNA unzips.
Canto: Well the base pairs are connected by hydrogen bonds, and the two DNA strands, the backbones of the molecule, run in opposite, or anti-parallel, directions, from the 5′ (five prime) end to the 3′ (three prime) end. So, while one strand runs from 5′ to 3′ (the sense strand), the other runs 3′ to 5′ (the antisense strand).
Jacinta: Right, so what we’re talking about here is DNA replication, which involves breaking those hydrogen bonds, among other things.
Canto: Yes, so that backbone, or double backbone whatever, where the strands run anti-parallel, is a phosphate-sugar construction, and the sugar is deoxyribose, a five-carbon sugar. This sugar is oriented in one strand from 5′ to 3′, that’s to say the 5′ carbon connects to a phosphate group at one end, while the 3′ carbon connects to a phosphate group at the other end, while in the other strand the sugar is oriented in the opposite direction.
Jacinta: Yes, and this is essential for replication. The protein called DNA polymerase should be introduced here, with thanks to Khan Academy. It adds nucleotides to the 3′ end to grow a DNA strand…
Canto: Yes, but I think that’s part of the zipping process rather than the unzipping… it’s all very complicated but we need to keep working on it…
Jacinta: Yes, according to Khan Academy, the first step in this replication is to unwind the tightly wound double helix, which occurs through the action of an enzyme called topoisomerase. We could probably do a heap of posts on each of these enzymes, and then some. Anyway, to over-simplify, topoisomerase acts on the DNA such that the hydrogen bonds between the nitrogenous bases can be broken by another enzyme called helicase.
Canto: And that’s when we get to add nucleotides. So we have the two split strands, one of which is a 3′ strand, now called the leading strand, the other a 5′ strand, called the lagging strand. Don’t ask.
Jacinta: The leading strand is the one you add nucleotides to, creating another strand going in the 5′ to 3′ direction. This apparently requires an RNA primer. Don’t ask. DNA primase provides this RNA primer, and once this has occurred, DNA polymerase can start adding nucleotides to the 3′ end, following the open zipper, so to speak.
Canto: The lagging strand is a bit more complex though, as you apparently can’t add nucleotides in that other direction, the 5′ direction, not with any polymerase no how. So, according to Khan, ‘biology’ adds primers (don’t ask) made up of several RNA nucleotides.
Jacinta: Again, according to Khan, the DNA primase, which works along the single strand, is responsible for adding these primers to the lagging strand so that the polymerase can work ‘backwards’ along that strand, adding nucleotides in the right, 3′, direction. So it’s called the lagging strand because it has to work through this more long, drawn-out process.
Canto: Yes, and apparently, this means that you have all these fragments of DNA, called Okazaki fragments. I’m not sure how that works…
Jacinta: Let’s devote our next post on this subject entirely to Okazaki fragments. That could clarify a lot. Or not.
Canto: Okay, let’s. Goody goody gumdrops. In any case, these fragments can be kind of sewn together using DNA ligase, presumably another miraculous enzyme. And the RNA becomes DNA. Don’t ask. I’m sure all will be revealed with further research and investigation.
References
Leading and lagging strands in DNA replication (Khan Academy video)