I have a main question that will lead to further questions depending upon the answer.

In the process of transcription, will there be multiple start and stop codons in one sequence of pre-mRNA? If there is, do they remain in the mRNA after splicing? If they do, does that mean one mRNA has the coding sequence for the synthesis of multiple different proteins?

One last question: assuming a single mRNA has the sequence for synthesis of a single protein, is the stop codon always the final sequence before the poly-A tail?

Thanks in advance for your help and sorry for the mini rabbit hole.

  • $\begingroup$ I've changed the title to reflect what seems to me the particular essence of the question: the concern with multiple start and stop codons. For this reason it doesn't really appear to be a duplicate. $\endgroup$
    – David
    May 25 '16 at 13:32
  • $\begingroup$ In reviewing some of my older answers in order to update them, I find this has been marked as a duplicate of a question that, at least in its current form it is certainly not a duplicate of. This question asks "does that mean one mRNA has the coding sequence for the synthesis of multiple different proteins?" The one it is a duplicate of only asks about stop codons. Is there a mechanism for rescinding this sort of incorrect decision? $\endgroup$
    – David
    Jul 11 '16 at 15:50

Whether or not there are multiple start and stop codons depends on what you mean by "start codon" and "stop codon".

The start codon has the sequence "AUG", and the stop codon has the sequence "UAG", "UAA", or "UGA". Both the pre-mRNA and the mature-mRNA can, and usually do, contain multiple instances of all of these sequences. However, only one "AUG" instance serves as the translational start site, and only one instance of the stop codon sequence serves as the translational stop site.

The translational start site is usually the first (5' most) AUG. However, for reasons that are still not entirely understood, in about 5% of genes the first AUG is skipped, and translation starts at one of the other AUG sequences.

The translational stop site is always the first stop codon to occur in-frame with the translational start site in the mature mRNA.

The stop codon is usually not (I'm tempted to say never) the final sequence before the poly-A-tail. The coding sequence (the RNA region that codes for the protein) occurs in the middle. There is a 5' untranslated region (UTR) before the protein coding region, and a 3' UTR after the protein coding region. The UTR sequences vary among genes and can have different functions depending on the sequence.

human mRNA diagram


As this question is a first post it is probably just a basic question about protein synthesis, which @Sean Johnson has answered adequately. However I’m not quite sure. And as I used to work in protein synthesis (but am a rather out of touch now) I decided to look at the recent literature a little to address some more esoteric or obscure questions it raised in my mind. I’ve set out the results below, and divided my answer into mature mRNA (which Sean covered primarily) but have also considered pre-mRNA. If the answer is not very useful to the questioner it may still be of interest to others.

Start and Stop Codons in Mature mRNA

The general picture of initiation and termination of protein biosynthesis in eukaryotes (bacteria and archea are different) has been described by Sean and can be found in standard texts1. In most cases the first AUG from the 5'-end of the mRNA is the one recognized as the ‘start’ signal for protein synthesis by the ribosomal-subunit initiation complex that scans along from the cap, according to the Kozak model2; and any one of the three termination codons encountered in the reading frame is recognized as ‘stop’ and (almost) invariably lead to termination of the polypeptide chain.

As mentioned, in some 5% of mRNAs certain aspects of the environment of the first AUG from the 5'-end results in it being ignored by the complex, and the insertion of methionine and start protein synthesis occurs at the second (or perhaps even a subsequent) AUG.2

Certain eukaryotic viruses were found to initiate the translation of the mRNAs internally, by a mechanism distinct from the 5'-scanning method. This involves an ‘internal ribosomal entry site’ — IRES. It subsequently transpired that a small subset of host mRNAs posses an IRES and can initiate translation internally. These include some growth factors, transcription factors and translation factors.3 Such (rare) mRNAs can therefore have several AUGs in their 5'-untranslated region.

Although it is possible to envisage an mRNA with several IRESs, and thus encoding several different proteins (analogous to a bacterial polycistronic mRNA), this does not appear to happen. It is striking, for example, that poliovirus RNA has just a single IRES for the start of the synthesis of single a large polyprotein, from which it generates multiple proteins by proteolysis. It would seem that the main purpose of this alternative initiation is to allow protein synthesis to proceed in the absence of a 5'-cap.

There are some AGU codons that are recognized by a specific tRNA that inserts selenocysteine into the polypeptide chain. Some aspect of the environment of these codons causes them to be ignored by the protein termination system, and the ribosome continues translating, eventually terminating at a subsequent stop codon4.

It has long been known that certain bacterial and eukaryotic viral mRNAs can increase their genetic repertoire by what is known as ‘read-through’ of ‘leaky’ stop codons. This is a ‘percentage’ effect, rather than all or nothing, and results in a mixture of ‘normal-length’ and extended products. It is now clear that the same phenomenon can occur in some eukaryotic mRNAs: quite frequently in Drosophila5, but also in mammals6.

I’m not clear what the questioner means by “is the stop codon always the final sequence before the poly-A tail?”. If the question is whether there can be additional stop codons in the 3'-UTR between the functional termination codon and the AATAAA polyadenylation signal (purple in Sean’s diagram) the answer is yes. Looking at the sequences of some muscle protein cDNAs done years ago, I found several stop codons in this region in each that I looked at. Of course they are without any function as the ribosome has already departed, but there is no reason for them not to be there on a random basis (and at apparently random positions).

Start and Stop Codons in pre-mRNA

By pre-mRNA I assume is meant the initial RNA transcript before splicing. There is no reason why an intron that is spliced out so that it does not appear in the final mRNA should not contain start or stop codons, which if not removed would have altered the reading frame. Many do.

A more interesting situation is where there is alternative splicing to produce different mRNA transcripts that encode different proteins. Pertinent to the question is the situation with start codons where the proteins differ in their N-termini, and stop codons where the proteins differ in their C-termini. The latter are quite common, but the former also occur, particularly were there are alternative forms of a protein, with and without a signal peptide7. In these cases the start or stop codons will be present in both of two exons, one of which is spliced out in each case. Examples of this from a couple of Drosophila genes are illustrated: Changes in N- and C-termini by alternative splicing

In both cases the coding regions are in orange, and the untranslated regions in grey. The thin lines represent the position of introns that have been spliced out. The arrowhead represents the direction of transcription and is at the C-terminal side of the protein. (i) Different N-termini for alternative products of gene vha14-2; (ii) Different C-termini for alternative products of gene vhaSFD.9


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