The death of the synonymous codon – V

The coding capacity of our genome continues to amaze. The redundancy of the genetic code has been put to yet another use. Depending on how much you know, skip the following four links and read on. Otherwise all the background you need to understand the following is in them.

https://luysii.wordpress.com/2011/05/03/the-death-of-the-synonymous-codon/

https://luysii.wordpress.com/2011/05/09/the-death-of-the-synonymous-codon-ii/

https://luysii.wordpress.com/2014/01/05/the-death-of-the-synonymous-codon-iii/

https://luysii.wordpress.com/2014/04/03/the-death-of-the-synonymous-codon-iv/

There really is no way around the redundancy producing synonymous codons. If you want to code for 20 different amino acids with only four choices at each position, two positions (4^2) won’t do. You need three positions, which gives you 64 possibilities (61 after the three stop codons are taken into account) and the redundancy that comes along with it. The previous links show how the redundant codons for some amino acids aren’t redundant at all but used to code for the speed of translation, or for exonic splicing enhancers and inhibitors. Different codons for the same amino acid can produce wildly different effects leaving the amino acid sequence of a given protein alone.

The latest example — https://www.pnas.org/content/117/40/24936 Proc. Natl. Acad. Sci. vol. 117 pp. 24936 – 24046 ‘2 — is even more impressive, as it implies that our genome may be coding for way more proteins than we thought.

The work concerns Mitochondrial DNA Polymerase Gamma (POLG), which is a hotspot for mutations (with over 200 known) 4 of which cause fairly rare neurologic diseases.

Normally translation of mRNA into protein begins with something called an initator codon (AUG) which codes for methionine. However in the case of POLG, a CUG triplet (not AUG) located in the 5′ leader of POLG messenger RNA (mRNA) initiates translation almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF) called  POLGARF (POLG Alternative Reading Frame — surely they could have come up something more euphonious).

Not only that but the reading frame is shifted down one (-1) meaning that the protein looks nothing like POLG, with a completely different amino acid composition. “We failed to find any significant similarity between POLGARF and other known or predicted proteins or any similarity with known structural motifs. It seems likely that POLGARF is an intrinsically disordered protein (IDP) with a remarkably high isoelectric point (pI =12.05 for a human protein).” They have no idea what POLGARF does.

Yet mammals make the protein. It gets more and more interesting because the CUG triplet is part of something called a MIR (Mammalian-wide Interspersed Repeat) which (based on comparative genomics with a lot of different animals), entered the POLG gene 135 million years ago.

Using the teleological reasoning typical of biology, POLGARF must be doing something useful, or it would have been mutated away, long ago.

The authors note that other mutations (even from one synonymous codon to another — hence the title of this post) could cause other diseases due to changes in POLGARF amino acid coding. So while different synonymous codons might code for the same amino acid in POLG, they probably code for something wildly different in POLGARF.

So the same segment of the genome is coding for two different proteins.

Is this a freak of nature? Hardly. We have over an estimated 368,000 mammalian interspersed repeats in our genome — https://en.wikipedia.org/wiki/Mammalian-wide_interspersed_repeat.

Could they be turning on transcription for other proteins that we hadn’t dreamed of. Algorithms looking for protein coding genes probably all look for AUG codons and then look for open reading frames following them.

As usual Shakespeare got there first “There are more things in heaven and earth, Horatio, Than are dreamt of in your philosophy.”

Certainly the paper of the year for intellectual interest and speculation.

The incredible information economy of frameshifting

Her fox and dog ate our pet rat

H erf oxa ndd oga teo urp etr at

He rfo xan ddo gat eou rpe tra t

The last two lines make no sense at all, but (neglecting the spaces) they have identical letter sequences.

Here are similar sequences of nucleotides making up the genetic code as transcribed into RNA

ATG CAT TAG CCG TAA GCC GTA GGA

TGC ATT AGC CGT AAG CCG TAG GA.

GCA TTA GCC TAA GCC GTA GGA ..

Again, in our genome there are no spaces between the triplets. But all the triplets you see are meaningful in the sense that they each code for one of the twenty amino acids (except for TAA which says stop). ATG codes for methionine (the purists will note that all the T’s should be U). I’m too lazy to look the rest up, but the ribosome doesn’t care, and will happily translate all 3 sequences into the sequential amino acids of a protein.

Both sets of sequences have undergone (reading) frame shifts.

A previous post https://luysii.wordpress.com/2014/10/13/the-bach-fugue-of-the-genome/ marveled about how something too small even to be called a virus coded for a protein whose amino acids were read in two different frames.

Frameshifting is used by viruses to get more mileage out of their genomes. Why? There is only so much DNA you can pack into the protein coat (capsids) of a virus.

[ Proc. Natl. Acad. Sci. vol. 111 pp. 14675 – 14680 ’14 ] Usually DNA density in cell nuclei or bacteria is 5 – 10% of volume. However, in viral capsids it is 55% of volume. The pressure inside the viral capsid can reach ten atmospheres. Ejection is therefore rapid (60,000 basepairs/second).

The AIDS virus (HIV1) relies on frame shifting of its genome to produce viable virus. The genes for two important proteins (gag and pol) have 240 nucleotides (80 amino acids) in common. Frameshifting occurs to allow the 240 nucleotides to be read by the cell’s ribosomes in two different frames (not at once). Granted that there are 61 3 nucleotide combinations to code for only 20 amino acids, so some redundancy is built in, but the 80 amino acids coded by the two frames are usually quite different.

That the gag and pol proteins function at all is miraculous.

The phenomenon is turning out to be more widespread. [ Proc. Natl. Acad. Sci. vol. 111 pp. E4342 – E4349 ’14 ] KSHV (Kaposi’s Sarcoma HerpesVirus) causes (what else?) Kaposi’s sarcoma, a tumor quite rare until people with AIDS started developing it (due to their lousy immune system being unable to contend with the virus). Open reading frame 73 (ORF73) codes for a major latency associated nuclear antigen 1 (LANA1). It has 3 domains a basic amino terminal region, an acidic central repeat region (divisible into CR1, CR2 and CR3) and another basic carboxy terminal region. LANA1 is involved in maintaning KSHV episomes, regulation of viral latency, transcriptional regulation of viral and cellular genes.

LANA1 is made of multiple high and lower molecular weight isoforms — e.g. a LANA ladder band pattern seen in immunoblotting.

This work shows that LANA1 (and also Epstein Barr Nuclear antigen 1` ) undergo highly efficient +1 and -2 programmed frameshifting, to generate previously undescribed alternative reading frame proteins in their repeat regions. Programmed frameshifting to generate multiple proteins from one RNA sequence can increase coding capacity, without increasing the size of the viral capsid.

The presence of similar repeat sequences in human genes (such as huntingtin — the defective gene in Huntington’s chorea) implies that we should look for frame shifting translation in ourselves as well as in viruses. In the case of mutant huntingtin frame shifting in the abnormally expanded CAG tracts rproduces proteins containing polyAlanine or polySerineArginine tracts.

Well G, A , T and C are the 1’s and 0’s of the way genetic information is stored in our genomic computer. It really isn’t surprising that the genome can be read in alternate frames. In the old days, textual information in bytes had parity bits to make sure the 1’s and 0’s were read in the correct frame. There is nothing like that in our genome (except for the 3 stop codons).

What is truly suprising it that reading in alternate frame produces ‘meaningful’ proteins. This gets us into philosophical waters. Clearly

Erf oxa ndd oga teo urp etr at

Rfo xan ddo gat eou rpe tra t

aren’t meaningful to us. Yet gag and pol are quite meaningful (even life and death meaningful) to the AIDS virus. So meaningful in the biologic sense, means able to function in the larger context of the cell. That really is the case for linguistic meaning. You have to know a lot about the world (and speak English) for the word cat to be meaningful to you. So meaning can never be defined by the word itself. Probably the same is true for concepts as well, but I’ll leave that to the philosophers, or any who choose to comment on this.