A synonymous codon that isn’t

Molecular biology is simply too elegant and beautiful to be left to the molecular biologists.  So I’m going to present the intriguing result of a recent paper as I would take notes on it for myself, and then unpack it explaining the various terms contained as I go along.

It you’re really adventurous — start reading a series of 5 posts I wrote starting with https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and follow the links.

It should explain everything in the paper below.

The paper itself is Nature vol. 602 pp. 335 – 342 ’22 — https://www.nature.com/articles/s41586-022-04451-4.pdf.

The unvarnished result:  Just mutating glutamine to lysine at position 61 of the KRAS oncogene (Q61K)isn’t enough to make KRAS resistant to an anticancer drug that attacks it (Osimertinib).  One of the synonymous codons for glycine at position 60 must be switched to another.

OK:  let’s unpack this starting with synonymous codon.

The DNA making up our genome is a string of elements (nucleotides also known as bases) strung together.  Similarly, our proteins are strings of elements (amino acids).  The order is crucial; just as it is with the 26 letters making up words. Consider the two words united and untied.

Bases come on 4 varieties (A, T, G and C).  Amino acids come in twenty varieties (of which three are glycine (G), Glutamine (Q) and lysine (K) — the one letter abbreviations don’t make much sense but that’s the way it is.

Since order of both bases and amino acids are important, it’s clear that  A T and T A are different. 2 bases  can only code for 16 amino acids.  Go up to 3 bases and you can code for 64 amino acids, which is overkill.   A sequence of 3 bases is called a codon. All 64 codons   code for an amino acid (except for three of them about which much more later).  This means that there must be several codons coding for the same amino acid —  these are the synonymous codons.

The number of codons for a given amino acid ranges from 1 (methionine M) to 6 (Leucine L).  Here are the 4 synonymous codons for glycine — GGA, GGC, GGG and GGT.  Note how similar they are.

Now the human genome has 3,200,000,000 bases strung together divided into 46 pieces (the chromosomes).  If placed end to end (Dorothy Parker fashion) they would be 3 feet 3 inches (1 meter) long.  All this is in a cell so small it is invisible to the naked eye.   If this is too much to get your head around, you might enjoy the following series of 6 posts — start here and follow the links https://luysii.wordpress.com/2010/03/22/the-cell-nucleus-and-its-dna-on-a-human-scale-i/

Any 3 bases linked together code for an amino acid, but there are many different ways to ‘read’ the genome. Among the many proteins our genome codes for are the transcription factors (1,639 of them as of 2018) which bind to stretches of 10 or more bases, to activate certain genes.   That’s 4^10 possibilities (over a million) allowing a unique binding site for the 1,639.  So transcription factors read the genome in groups of 10 or so not 3.

There is yet another way to read the genome, and this has to do with the fact the genes coding for proteins are much longer (have more bases) than the 3 times the number of amino acids they code for.  The classic example is dystrophin, a gene mutated in Duchenne muscular dystrophy.  It’s a monster protein with 3,685 amino acids — so it needs 3,685 *3 = 11,055 bases in a row to code for them at 3 bases/amino acids.  The dystrophin gene, however, stretches for 2,220,223  bases.  So the protein coding parts of the gene (the exons) come in 79 different pieces separated by parts that don’t code for amino acids (the introns).

I’m skipping a lot here, but the introns must be spliced out of a copy of the gene (mRNA).  Again the genome is read by yet another machine (the spliceosome) which removes introns from newly formed copies of the gene (the mRNA).  The spliceosome is a huge molecular machine containing 5 RNAs (called small nuclear RNAs, aka snRNAs), along with 50 or so proteins with a total molecular mass again of around 2,500,000 kiloDaltons (a carbon atom is 12 Daltons).  Most proteins have introns and exons, and most of them exist in multiple forms due to alternative splicing of introns.  The spliceosome reads the mRNA in 6 – 8 base chunks looking for sites (splicing sites) to bind and begin splicing out introns. Yet another way to ‘read’ a sequence of bases.   Exon sequences which promote or repress alternative splicing sites are known (these are called EXE == exonic splicing enhancers, and ESSs = exonic splicing suppressors).

And now, at very long last, we get to the four synonymous codons of glycine which aren’t functionally synonymous at all.  This isn’t trivial: they determine the base sequence a mutated gene must have to produce cancer.

Here’s the unvarnished result once again — Just mutating glutamine to lysine at position 61 of the KRAS oncogene (Q61K) isn’t enough to make KRAS resistant to an anticancer drug that attacks it (Osimertinib).  One of the synonymous codons for glycine at position 60 must be switched to another.

What is KRAS?  A protein which gets its name from a virus causing cancer in rats.  Kirsten RAt Sarcoma virus.  KRAS, when active, relays signals from outside the cell to the nucleus to make the cell proliferate.  The protein exists in active and inactive forms.  Humans have KRAS, and 3 similar proteins.  Mutations causing  members of the protein family to remain in constantly active form are found in 1/3 of all cancers.  In the case of KRAS some activating mutations occur at positions 60 and 61 of the 189 amino acid protein.  That’s all it takes.

The codon for glutamine at position 61 in KRAS is CAA.  To change it to the codon for lysine requires a change of just one base e.g. from CAA (glutamine) to AAA (lysine) and now you have  a KRAS which is always active producing cancer.

Recall that glycine has 4 codons (GGA, GGC, GGG and GGT).  The one found in unmutated KRAS is GGT.  This codon is never found in the KRAS Q61K mutant seen in tumors.  Why?  Because GGTAAA forms a splice site which the splicing machine uses to cut out a different set of introns going to an exon.  This exon contains one of the 3 codons  mentioned above not coding for an amino acid.  They are called termination codons or stop codons, and tell the machinery making mRNA from DNA to quit.   This means that the full mutated  KRAS with its 188 amino acids is never made.  So tumor producing KRAS has GGGAAA or GGAAAA or GGCAAA at positions 60 and 61 and never GGTAAA

So the 3 synonymous glycine codons have very nonsynonymous effects.  Now you know.  Elegant isn’t it?

 

 

I sincerely hope it works, but I’m very doubtful

A fascinating series of papers offers hope (in the form of a small molecule) for the truly horrible Werdnig Hoffman disease which basically kills infants by destroying neurons in their spinal cord. For why this is especially poignant for me, see the end of the post.

First some background:

Our genes occur in pieces. Dystrophin is the protein mutated in the commonest form of muscular dystrophy. The gene for it is 2,220,233 nucleotides long but the dystrophin contains ‘only’ 3685 amino acids, not the 770,000+ amino acids the gene could specify. What happens? The whole gene is transcribed into an RNA of this enormous length, then 78 distinct segments of RNA (called introns) are removed by a gigantic multimegadalton machine called the spliceosome, and the 79 segments actually coding for amino acids (these are the exons) are linked together and the RNA sent on its way.

All this was unknown in the 70s and early 80s when I was running a muscular dystrophy clininc and taking care of these kids. Looking back, it’s miraculous that more of us don’t have muscular dystrophy; there is so much that can go wrong with a gene this size, let along transcribing and correctly splicing it to produce a functional protein.

One final complication — alternate splicing. The spliceosome removes introns and splices the exons together. But sometimes exons are skipped or one of several exons is used at a particular point in a protein. So one gene can make more than one protein. The record holder is something called the Dscam gene in the fruitfly which can make over 38,000 different proteins by alternate splicing.

There is nothing worse than watching an infant waste away and die. That’s what Werdnig Hoffmann disease is like, and I saw one or two cases during my years at the clinic. It is also called infantile spinal muscular atrophy. We all have two genes for the same crucial protein (called unimaginatively SMN). Kids who have the disease have mutations in one of the two genes (called SMN1) Why isn’t the other gene protective? It codes for the same sequence of amino acids (but using different synonymous codons). What goes wrong?

[ Proc. Natl. Acad. Sci. vol. 97 pp. 9618 – 9623 ’00 ] Why is SMN2 (the centromeric copy (e.g. the copy closest to the middle of the chromosome) which is normal in most patients) not protective? It has a single translationally silent nucleotide difference from SMN1 in exon 7 (e.g. the difference doesn’t change amino acid coded for). This disrupts an exonic splicing enhancer and causes exon 7 skipping leading to abundant production of a shorter isoform (SMN2delta7). Thus even though both genes code for the same protein, only SMN1 actually makes the full protein.

Intellectually fascinating but ghastly to watch.

This brings us to the current papers [ Science vol. 345 pp. 624 – 625, 688 – 693 ’14 ].

More background. The molecular machine which removes the introns is called the spliceosome. It’s huge, containing 5 RNAs (called small nuclear RNAs, aka snRNAs), along with 50 or so proteins with a total molecular mass again of around 2,500,000 kiloDaltons. Think about it chemists. Design 50 proteins and 5 RNAs with probably 200,000+ atoms so they all come together forming a machine to operate on other monster molecules — such as the mRNA for Dystrophin alluded to earlier. Hard for me to believe this arose by chance, but current opinion has it that way.

Splicing out introns is a tricky process which is still being worked on. Mistakes are easy to make, and different tissues will splice the same pre-mRNA in different ways. All this happens in the nucleus before the mRNA is shipped outside where the ribosome can get at it.

The papers describe a small molecule which acts on the spliceosome to increase the inclusion of SMN2 exon 7. It does appear to work in patient cells and mouse models of the disease, even reversing weakness.

Why am I skeptical? Because just about every protein we make is spliced (except histones), and any molecule altering the splicing machinery seems almost certain to produce effects on many genes, not just SMN2. If it really works, these guys should get a Nobel.

Why does the paper grip me so. I watched the beautiful infant daughter of a cop and a nurse die of it 30 – 40 years ago. Even with all the degrees, all the training I was no better for the baby than my immigrant grandmother dispensing emotional chicken soup from her dry goods store (she only had a 4th grade education). Fortunately, the couple took the 25% risk of another child with WH and produced a healthy infant a few years later.

A second reason — a beautiful baby grandaughter came into our world 24 hours ago.

Poets and religious types may intuit how miraculous our existence is, but the study of molecular biology proves it (to me at least).