Covid-19: A Primer On DNA, RNA, and SARS-CoV-2 January 13, 2021

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I want to explain a few things about SARS-CoV-2 (the virus that causes Covid-19) and the vaccines that are being rolled out in both the UK and the world. To do so I first need to explain about DNA, RNA, proteins and what is called the central dogma of molecular biology, which is what this post is about. The central dogma is the core – the absolute fundamental key thing of life, of our biology. It is the biological equivalent of what quantum mechanics is to physics.

Thankfully, it is far simpler to understand the basics of the central dogma of molecular biology than the basics of quantum mechanics. It is also a well established concept, I was taught it last century at university and it has not changed much in the 32 years or so, though we better understand so much more of the details and ramifications now (6).

Central Dogma Of Molecular Biology

Basically the Central Dogma is that DNA makes RNA and RNA makes protein – and information does not flow backwards. I’ll try and explain that in steps, but before that I want to give a quick reminder about DNA, which most of you probably remember from school, and protein/polypeptides. Sorry, but it’s necessary. Skip to “The Core Of Biology” if you already know all about these.

DNA and Proteins

DNA molecule – from Yourgenome.org

All living organism contain and are controlled by DNA – Deoxyribonucleic Acid. This is the helical, double-stranded molecule whose structure was worked out by Watson, Crick, and Rosalind Franklin. In all organisms (except bacteria & archaea – together known as prokaryotes) the DNA is held in the nucleus of the cell (1). The whole genome is in every normal cell in an organism (be it a plant, fungus, moss, animal, you. Everything alive that is not bacteria/archaea is a eukaryote – which means the cell has a nucleus). There are some exceptions – such as red blood cells that lack a nucleus, or sex cells that carry one half of the normal amount of DNA for a given species.

The DNA directs all of the biochemistry of an organism (2). Everything. It defines the structure of the proteins we are made of, how the proteins go together, the layers and parts of our organs, the overall plan of our bodies, and the hormones, chemicals, and free-moving cells that go around our bodies like red and white blood cells. We still don’t know how some of this control is done but as DNA changes (mutations) affect all of these things, we know the DNA is basically the instruction book to both make an organism and to keep it functioning.

The DNA of an organism contains (amongst other things) it’s genes. Genes are the instructions for making all of our proteins and we say the genes are “expressed” when the genes are activated and make the proteins. And they do this all the time, at great rates, churning out vast quantities of proteins in each and every cell.

I say proteins but that is not *quite* right. Proteins are made of polypeptides, and polypeptides are made up of amino acids. As an analogy, proteins are paragraphs, polypeptides are sentences, sentences are made of letters which are the amino acids.

In the vast majority of organisms there are 20 different amino acids available to “spell out” all polypeptides. Genes have instructions to make chains of amino acids, called polypeptides. A protein may be a polypeptide, but it might also be made of several polypeptides or polypeptides that have been chemically modified after being initially made. I make this point as below, and in some of the links, polypeptides and amino acids are referenced and general scientific literature can be a bit muddled between polypeptides and proteins. For now, just think of proteins as really complex polypeptides. (3)

DNA consists of four letters as you probably know. Adenine, Cytosine, Guanine, and Thymine. In the double helix the two strands are “inverse mirror images” of each other and the letters pair up – A with T and C with G. So a short strand of DNA might be something like the below and, as indicated, the two halves can be split and, via the pairing of the letters, perfectly copied:

Double stranded DNA can be split and (perfectly) duplicated

The DNA is unzipped and split (by an enzyme called Helicase) and then DNA Polymerase can come along and add in the missing letters and you end up with two perfect copies. Usually. In my example, to the right, there is a mistake,  – a C has been added in where an A should be. This mistake is an example of a mutation.

However, that is not part of the Central Dogma. Although DNA duplication is vital (after all, every cell needs a full copy of the organism’s genome so the DNA needs to be replicated each time the cell divides). The main function of DNA is to pour out instructions for the creation of polypeptides

Central Dogma by Brownfield 5 on slideshare

The Core of all Biology

All the complexity of what our cells do and how our biochemistry works is via genes being expressed.  I’m not going to even attempt to describe how gene expression is controlled. It’s incredibly complex, it’s an area of understanding that has advanced hugely since I was taught the basics in my degree 32 years ago, and science still does not really understand a lot of it. But it’s the expression of these genes that allow are cells to do what they do, from growing hair, muscles, and making white blood cells to producing the enzymes that digest our food and control our bodies. All cells express thousands of genes all the time, at different levels of expression, and they do this by producing Messenger RNA, known simply as mRNA.

RNA, or Ribonucleic Acid, is (as the name implies) structurally very similar to DNA. It is single stranded, not double stranded like our genomic DNA, and the Thymine is replaced with a very similar chemical called Uracil.

Transcription

When a gene is expressed the relevant piece of DNA is “unzipped” and an enzyme called RNA Polymerase walks along the DNA and creates a complementary (meaning A becomes U, T become A, C becomes G and G becomes C) RNA copy of the DNA, as is shown in the diagram to the right. This is called transcription and it produces something called pre-mRNA. This is itself then processed by by other enzymes which cut out parts of the RNA that represent “Introns” – bits of DNA in the gene that are not to be used. This bit is not shown on the diagram. It is one of the complexities of our DNA that was poorly understood before the Human Genome Project and is still rarely explained. In a complex organism like a mammal or plant or fish, a single gene can produce a range of proteins depending on how this pre-mRNA is processed.

The diagram below shows how the double-stranded DNA is “unzipped” and the RNA polymerase reads one strand of the DNA and produces an RNA strand based on it.

RNA Polymerase, from BC Opentextbooks

The final mRNA consists of three main parts. The first part, at what is called the 5 prime (or 5′) end is a cap that allows the mRNA to be recognised and grabbed by the Ribosomes (see later) and transcribed. Then comes the RNA equivalent of the DNA gene for the protein. At the other end, the 3 prime (or 3′) end, a string of As is added (100-200 of them normally), the poly-A tail. We will see why later.

Pre-mRNA to mRNA from Wikipedia

This mature mRNA is then transported out of the nucleus of the cell and out into the body of the cell, into the cytoplasm. The mRNA may hold markers to say where specifically in the cell it is to go but that’s a detail I won’t go into. This seems to be very important to complex organisms like ourselves, that the DNA sits in the nucleus, mRNA is produced and this is quickly passed out of the nucleus to be processed elsewhere in the cell.

Translation

So we now have an mRNA molecule in the cell ready to be translated, i.e. used to make a polypeptide. In the cell there are thousands and thousands of very complex molecules called “Ribosomes”. A ribosome is actually made of two parts, both of which are themselves made out of RNA, not protein. These ribosomes clamp onto the cap of the mRNA, one part on one side, the other on the opposite side, the mRNA in the middle. The cap is a special starter molecule and the first part of the mRNA, which does not code for a polypeptide but controls how easily the Ribosome attaches to the mRNA. The attached ribosome will then “read” the mRNA, working down the string of letters and creating a polypeptide that is described by the RNA sequence.

Special intermediate molecules are used to translate the RNA to an amino acid. These are the tRNA molecules shown in the diagram below. As a ribosome walks along the mRNA is reads small chunks of the mRNA, finds a tRNA that complements the mRNA and temporarily binds it to the mRNA. At the other end of the tRNA is a specific amino acid and this bonds to the growing polypeptide/protein.

Ribosome translating mRNA – from sites.google.com

Many ribosomes can be walking along a single mRNA molecule  at a time, creating more than one copy of the polypeptide. When a Ribosome gets to the end of the mRNA, to the poly-a tail, it drops off (4) and it will (or at least may) snip off the very end of the poly-a tail. So it shortens the tail. This tail protects the mRNA molecule from being destroyed by the cell so, as it shortens then the mRNA becomes more likely to be destroyed. Why is this important? Well, the cell needs to produce different polypeptides at different concentrations at different times. The poly-a tail is a key part of how the cell controls how long an mRNA molecule last for, creating polypeptides. The longer the tail, the longer the mRNA lasts. The mRNA can’t be allowed to hang about in the cell producing polypeptide for ever. Once it’s tail is gone it is destroyed. Thus for a polypeptide to be constantly produced, the genes in the nucleus need to keep producing the mRNA for it.

As you can appreciate, changes to the 5′ cap can change how quickly (and often) a ribosome latches onto an mRNA molecule, the length of the poly-a tail can control how long the mRNA lasts and so how much polypeptide that one mRNA molecule creates, and the nucleus has overall control over when and how much mRNA is produced. These processes allow constant control and change to how a particular gene is expressed.

I’ve said about the RNA being read and converted into a polypeptide. How does this work? The diagram above and the tRNA give clues to this.

How Codons translate to amino acids

As I said earlier, there are four letters of the RNA alphabet – A,C,G, and U – and a polypeptide is like a sentence. There are 20 types of amino acids that are strung together to make our polypeptides. Our genes are written in sets of 3 letters, called codons. You can think of them as a gene “word”. You can see this with the tRNA molecules in the diagram, at one end they have three RNA letters, and the other end the specific amino acid that those three RNA letters translate to.

A codon, such as UCA, codes for an amino acid called serine (or Ser). GCU codes for alanine (Ala). With 4 letters and a “word” being 3 letters long, there are 64 combinations of A,C,G, & U possible. You can see all 64 of these combinations in the table to the left. So how do the 64 possible codons map to 20 amino acids? Well, some codons have a special meaning.

3 – UAA, UAG, & UGA – are stop codons. They mean “this polypeptide is finished, ribosome stop reading”. One codon is special, AUG. This either means “start reading here” (note, there have to be other sequences in the RNA near it to make it mean this) or amino acid methionine (Met).

As for the others, well several codons mean the same amino acid, as you can see in the table to the left (5). Generally the first two letter in the codon define which amino acid the codon is for (anything starting GU is for valine, Val) but for some the third letter is the deciding factor.

Ribosomes thus start at a special AUG codon on the mRNA and then read three letters at a time and for each one, for each codon, the specific amino acid is added to the growing chain – via the tRNA molecules. The chain can be thousands of amino acids long or just a handful. The chain grows until a stop codon is reached. The longest polypeptide is titin, which is between 27,000-35,000 amino acids long (due to those “introns” I mentioned, sometimes some are cut out, sometimes not) and makes muscle elastic.

That’s it. That is how our DNA, our genes, make all the things that build and control our bodies. Of course, there is an incredible amount of complexity that arises from that central process, like how do the proteins control the inclusion of calcium to make our bones, grab iron and put it in our blood, and stuff all that fat in our cells. But it is all controlled and mediated through polypeptides, through proteins and enzymes.

If you want to see a large version of the Central Dogma diagram you can click here:

->ci350poster-141201170705-conversion-gate02

mRNA vaccines.

I won’t go into too many details here, but you may know that a couple of the Covid-19 vaccines are mRNA based. These vaccines are millions of mRNA molecules packaged up into little balls of fat (also called lipid). These mRNA molecules are all the same and are the instructions to produce the spike protein of Covid-19, the bit the virus uses to attach our cells and that the immune system is good at identifying and attacks. This is the Moderna and Pfizer vaccines

The vaccines are ONLY for the spike protein, not the rest of the virus, so the vaccines cannot give you Covid-19. But what the mRNA does do is get into your cells and your cell ribosomes latch on to the mRNA and make the spike protein. The mRNA molecules is not exactly like the one produced by the virus, it is modified to be more stable and last in the cell longer, with for example a longer poly-A tail. The longer it last, the more spike protein is produced. The vaccine mRNA is engineered to produce as much spike protein as possible.

Your body sees this spike protein, it knows it is “foreign” to your body and learns to attack it. Then, if you are infected by the real virus, your body will already know to attack the spike protein and either you do not get ill or you get less ill. Fantastic, isn’t it? I love science and I especially love biology and medicine. Even just 50 years ago this virus would have had to run it’s natural course through us and kill maybe like Spanish ‘flu did, but now we understand so much better what is going on and we can now do something about it. I say “we”, I mean biomedical scientists.

Previous vaccines have relied on getting a modified or damaged version of the whole virus into your body, or modifying another virus that is harmless so that it produces mRNA for exact copies of parts of the dangerous virus. These are hard things to do. The Oxford vaccine is a modified virus (a chimpanzee adenovirus, chosen I believe because it can infect us but does no harm and does not spread in humans).

Advances in handling mRNA, creating it, and understanding how to make it work in our cells, have allowed scientists to create mRNA vaccines which are simpler, more efficient, easy to create or modify , and more targeted than traditional vaccines. Only part of the virus is made and nothing else, so there is no danger whatsoever of the vaccine causing the disease, or a modified version of the disease. Further, if the virus alters (something that is a current worry) then modifying an mRNA vaccine is theoretically very easy and quick. Testing the new version would be necessary and would take months (regulatory bodies allowing – they might let a modified version be fast-tracked, my wife is an expert in pharmacovigilance and she thinks it could be done) , but it means mutations to SARS-CoV-2 can be handled relatively quickly if the need arises. This could be vitally important.

This work has not all been done since Covid-19 appeared about a year ago, it is based on several years of work on MERS, SARS and other viruses. So on the one hand these mRNA vaccines are a new technology, but they are new since 4 or 5 years ago and great advances in how to create them have been made in the last year.

Implications of the Central Dogma

Some things follow on from the above that are fundamental to SARS-CoV-2 and vaccines. I’ll touch on them here and expand on them in further posts, as this is a lot in one go.

Mutations and Open Reading Frame

In my previous post on the new variant of SARS-CoV-2 I mention mutations. At the start of this post I mentioned the copying of DNA/RNA and how mistakes can be made, in particular a single letter changing to another. When a single letter of the genome gets changed, this is called a Single Nucleotide Polymorphism or SNP. If that letter is the first one in a codon in a gene, it is almost certainly going to have an impact. It will change the amino acid inserted at that point in the polypeptide. If you look at the codon table earlier it is possible to change the first letter A to C and still get Arginine. Other than that, altering that first letter in the codon alters the polypeptide. Other SNPs can have no effect – changing GUU to GUC still makes valine. Of course, any SNP that creates a stop codon is going to have a potentially massive effect.

SNPs that cause no change to the polypeptide are called synonymous. As they make no difference to the organism, they occur and get passed to the next generation of the organism and all their offspring. We can use these synonymous SNPs to track the lineage of organisms and they are used to track the lineage of SARS-CoV-2. This allows us to, for example, track how the virus has spread geographically. I say us, I mean phylogenetic scientists.

Those SNPs that change the polypeptide sequence are more likely to change something about the biology of the organism. If the change is negative (for example it reduces the efficiency of an enzyme) the organism and it’s descendants will be at a disadvantage and the change will be selected out. If it gives the organism an advantage, it and it’s descendants will do better than those without the change and will take over in the population. This is true of the new variant B.1.1.7 – is better at spreading, it is taking over. Many SNPs that change one amino acid have very little positive or negative effect on the virus. (I don’t know what it is like on modern genetics degree courses but in my day lecturers would almost come to blows over how much effect a single mutation would need to have to be significant, and how much evolution of DNA was just mathematical, accidental drift, and how much was through selection pressure.)

Other, rarer mutations can be deletions and insertions. Extra letters get added or removed. Now, if the number of letters added/removed is 1, 2, 4, 5 or any number that is not divisible by 3, the impact is huge. Why? Well, a gene has something called it’s Open Reading Frame. Codons are always 3 letter long, staring at the ALU that initiates the mRNA being read. That reading frame of 3 letters per word has to be preserved through the whole gene. If you shift all the letters along by anything other than a multiple of 3, everything after that change becomes very different – and usually garbage.

An insertion or deletion of a non-multiple of 3 letters will not be significant if it occurs in DNA/RNA that does not code for something, but if it is in a gene it is 99.9837% (2) of the time a disaster for that gene, destroying the function of that polypeptide it producers. SARS-Cov-2 is an RNA virus and such viruses are almost all functional gene. Thus deletions or insertions that do not preserve the reading frame are rare (but do occur) in SARS-Cov-2 and other viruses.

Variant B.1.1.7 has 3 deletion mutations in it but they all preserve the reading frame. They drop 1,2, or 3 amino acids out of the polypeptides they code for. But the rest of the polypeptide is preserved. One particular deletion, in the spike protein removing amino acids 69 and 70, stops one of the standard PCR tests from detecting RNA fragments of SARS-CoV-2. I’ll revisit this topic in another post, but because it stops one of the standard PCR tests from working, that can be used in many situations for tracking this variant. Don’t worry, PCR tests for SARS-CoV-2 use 2 or 3 RNA sites to identify the virus, so it is still detected. However, the failure of one of the “channels” has turned out to be a boon for tracking this nasty variant.

Single Direction Of Information.

Under the central dogma you will see that information flows from DNA in the cell nucleus, to mRNA that leaves the nucleus and goes into the cell cytoplasm, and this is translated into polypeptides. It does not go the other way.

Nothing I know of in biology can take a polypeptide, let alone a mature protein, and generate RNA from it. Nothing. Humans can make a stab at it, we can look at the amino acid sequence of a protein and design an mRNA strand that might sort-of work but it’s hellishly difficult as organisms like terrestrial plants and vertebrates have complex post-processing of many polypeptides. Biology cannot do it.

There is nothing that takes mRNA and pulls it back into the cell nucleus and shoves it into our DNA. Nothing natural can do this that I am aware of. A couple of people on a social media forum full of biology experts that I mentioned this post on have voiced possibilities, but nothing concrete yet has been forthcoming (and it would be fascinating to learn about if they do, I’m always looking to learn).

Some of you may have heard of retroviruses such as HIV (the virus that causes AIDS). They can do something that sounds similar – but it is not. They can reverse transcribe their own RNA, i.e. create a DNA copy of their RNA using a reverse transcriptase enzyme, and insert it into the host DNA using an integrase enzyme. The retrovirus has the RNA genes for these two proteins in it’s genome, it brings it’s tools with it. They get into the nucleus of the host cell and use their own tools to insert their own genome into the host, along with control DNA so that the viral DNA can be expressed. What it does NOT do (as far as I know and this is possible where I am wrong) is grab random mRNA from around it and insert it into the DNA of the host. Remember, mRNA is exported out of the nucleus. It’s not there in the nucleus, at least not for long. Also, even if a retrovirus was to insert mRNA into the host’s DNA, it would be doing so without promoter sequences and all the stuff needed to get a gene to be expressed.

I make this point as some people on social media have claimed the mRNA in Covid-19 vaccines could get into your DNA. No, it can’t. It won’t. Anyone claiming this does happen does not understand the central dogma of molecular biology. Either that or they could be in line for a Nobel Prize in biology.

Firstly, the vaccine does not get into the nucleus. Second, there is no biological process native to vertebrates to do the insertion of mRNA into DNA. Third, even if by some chance a virus like HIV was present, and by some miracle some of that mRNA for the vaccine got into the nucleus, HIV is inserting a copy it’s own DNA, not random mRNA hanging around. Finally, even if a miracle on a miracle occurred and the mRNA from the vaccine was inserted into your DNA – there would be nothing to cause it to be expressed. It would just sit there doing absolutely nothing.

What Retroviruses can do is insert into a DNA genome, then when it is expressed it can occasionally pick up DNA from around where it inserted into the genome, which is transcribed and included into the RNA for the virus. If this modified virus then infects another organism and takes that original host DNA (as an RNA copy) with it, it can then insert that picked-up DNA into the new host. It’s very rare, it can happen. But no reverse reading of mRNA was involved.

Basically, the idea of mRNA from a vaccine getting into your genome is damned close to impossible given the current understanding of molecular biology.

Viruses

I’ll finish with some information on viruses.

Viruses are weird. There is an ongoing debate (and has been for over 35 years, as it was a topic of discussion during my degree) whether viruses are alive. Viruses can’t do anything without a cell and it’s machinery to make proteins from DNA/RNA. They can’t move themselves, they get moved about by mechanical processes (in droplets of liquid, floating in water, blown around in the air, transferred via fluids in real living things…). They don’t grow, they do not respond to stimuli (all other life from bacteria up do). They do nothing. A virus consists of just a few things:

• A string of genetic material, either double stranded DNA similar to what is in us, single stranded DNA or RNA (usually single stranded but occasionally double stranded). SARS-CoV-2 is a single stranded RNA virus.
• A protein coat, called a capsid, encapsulating the genetic material, keeping it protected and whole. This might be a simple, uniform coat or something more complex made of many proteins. SARS-CoV-2 has a capsid made of several proteins including the famous “spike” protein, which sticks out of the capsid and is what latches onto the ACE2 proteins on our cell walls and allows the virus to get into the host cell.  In SARS-CoV-2 these proteins are embedded in a lipid bilayer (a 2-molecule wide sphere of fat molecules). This is why simple soap is so effective at destroying the virus – it breaks up the lipid bilayer.
• Protein(s) within the capsid, binding to and protecting the genetic material. SARS-CoV-2 has this.
• A virus may have an outer lipid (fat) layer, usually derived from the lipid layer of the host cell it infected. SARS-CoV-2 does not have this. {and I might have slightly misunderstood whether this is an extra layer over and above the lipid bilayer that the protein coat can incorportate)

I think of viruses as very, very complex poisons and not alive. Others think of it as alive.

If you want to know more about the structure of SARS-CoV-2 this paper on the structure of the virus on the NCBI site is very good but quite technical.

The genetic material for a standard virus (like SARS-Cov-2) codes for a load of mRNAs that usurp the polypeptide making machinery of the host cells. i.e., they use the second half of the central dogma. Once the virus gets into a host cell, the mRNA is released and it hijacks our own cell’s ribosomes. It makes new proteins to make the virus shell and proteins to coat the RNA of the virus. It creates an RNA Polymerase enzyme to replicate it’s own genetic code and, in the case of coronaviruses like SARS-CoV-2 (and other types of virus) it produces a “checking enzyme” to make sure the RNA copies accurately.

This last point is very interesting. All organisms mutate but RNA viruses are the fastest mutating thing we know of. But SARS-CoV-2 mutates slowly for an RNA virus as it has a check enzyme. That’s one thing to be very thankful for. Influenza is an RNA virus that does not have a checking enzyme, which is part of why it changes so quickly and we need a new vaccine for it each year.

All these bits of the virus then self-assemble into thousands of new copies of the virus, burst the host cell and go and infect other cells in the organism. Some are ejected from the host organism in droplets coughed out or similar mechanical processes and infect other hosts.

That’s pretty much all that a virus does.

Notes

1) I said all our DNA is in the nucleus and controls everything. This is not quite true and I am sure some of you know that. We also have DNA in our mitochondria, the organelles in our cells providing us with energy at a biochemical level. Mitochondria look a little like bacterial cells living within our cells and some scientists think this is where they originally came from. It is suggested that a very early Eukaryotic cell absorbed and made a symbiotic relationship with a bacterial cell that was very good at making ATP (the unit of energy in most biology). This was so successful that the organism that did that out-competed all other Eukaryotic life and took over. And, over time the absorbed bacteria became simplified and specialised as the mitochondria. As a result, mitochondria have their own DNA. As do chloroplasts in plants.

2) There is really only one hard, absolute rule in biology. There is an exception to every absolute rule. See 1! Forgive me if I don’t cover all the exceptions in the rest of this post, but what I sat here is true 99.9837% of the time. And treat all percentages in documents with scepticism, many are made up.

3) The distinction between amino acids and peptides has always annoyed me. If “Amino acid” is the term for the building blocks of proteins should not a chain of amino acids be a “polyamino” or something? No, we have peptides/polypeptides.  A peptide has to be 2 or more amino acids as it is named after the bond between the two amino acids. A Peptide bond. Strictly speaking a peptide of between 2 and 20 amino acids is called an oligopeptide, and above that is a polypeptide. It’s just messy.

4) The ribosome may not drop off the mRNA. If the poly-A tail and the mRNA cap are intact, they my bind together to form a loop that allows most of the ribosomes to simply circle around the whole mRNA and make more polypeptide more efficiently. This might help curtail the activity of the mRNA more quickly as, when the poly-A tail or the cap are degraded (as there is some mechanism to degrade the cap too), then the loop is broken and Ribosomes can no longer cycle around. I don’t know the details.

5) The codon to amino acid mapping is very nearly universal. Almost all organism use the same mapping and it is one of the proofs that all life on this planet is related. However, there are some exceptions (as there always are in biology). If you want to nerd out on it look at this Wikipedia page on alternative codon translation tables.

6) The basics of the central dogma of molecular biology has been known for over 50 years. Here’s the start of the chapter on it from my 33 year old  “Genes 3” by Benjamin Lewin. Looking back at this book, which I pretty much knew cover to cover back then, I realise how much knowledge has leaked out my head.

This is the central dogma in my genetics undergraduate text book from 1988