Post-Transcriptional Modification

Guess who it is again? You guessed right, it’s me! I’d apologize for the “biochemistry” [cough genetics cough] spam, but an apology implies you’ll try not to do something again, and, wellllll, let’s just say the night is young. Very young.

I’ve got half a Mountain Dew, so let’s dive in before I run out!

In prokaryotic organisms (I’m looking at you, bacteria), once you’ve got an mRNA transcript, you’re gravy. All you have to do is shove that sucker through a ribosome, and you’ve got a lovely protein in the same compartment as the compartment in which you made the transcript in the first place.

However, unfortunately for biochemistry students, eukaryotes have, well, nuclei. (In fact, they’re named for them.) Because of that, we have to make the mRNA transcript in the nucleus (which houses the DNA), then shuttle that sucker into the cytosol. Along the way, eukaryotes pull eukaryotic shenanigans. That is, they flex their muscles of complexity and edit those punks.

Now, the thing to keep in mind about eukaryotic transcripts is that, unlike prokaryotic transcripts, they’re composed of introns and exons. Introns are noncoding regions of the transcript that space out coding regions called exons. (In fact, my book says that most of the stuff in introns is untranslatable nonsense.) Lengths of exons generally don’t vary between genes in different species, but the lengths of introns can vary dramatically.

Obviously, we need to get rid of all of that genetic nonsense punctuating the actual usable bits of our mRNA. However, before we do that, we have to carry out a couple of other modifications.

First, we need to add something called a 5′ cap to our transcript. This cap serves a lot of purposes, according to Wikipedia, but the one that we emphasized is that it keeps the mRNA from getting degraded. Our cells do this in a rather simple manner: an enzyme called guanyl transferase transfers the first phosphate group of GTP to the second of the 5′ nucleotide in the mRNA. This thing is then methylated on the 7th position of the guanine, and sometimes on the 2’OH groups of following two nucleosides or the 6-amino group of the first adenine.

Next, we add a poly-A tail. Most mRNA contain a consensus sequence toward their end (AAUAAA) called the “polyadenylation signal.” Ten to thirty nucleotides downstream from this sequence, exonucleases cleave off the end of the mRNA. Poly(A) polymerase then strings hundreds of adenines onto the end of the transcript. This also helps keep it from getting chewed up out there in the cytoplasm, as well as helping with other things.

Finally, before our mRNA leaves the nucleus, we have to splice out those introns. This has to be done very specifically—take one too few or too many nucleotides, and you screw up the whole transcript. Thankfully, introns are marked pretty well, with a 5′-GU and a 3′-AG (I remember them as “GUHH!” and “AAGH!” when I’m studying them) at either end. These, along with internal splice sites, help the introns get removed properly.

In fact, the mechanism for this isn’t too inaccessible. The 5’G of the intron is looped around and bonded to the 2’OH of the A in the branch site, forming a weird branched nucleotide structure. The 3’OH of a G at the end of the exon then bonds to the 5′-phosphate of the nucleotide at the beginning of the next exon. The intron then floats sadly away, awaiting its inevitably rapid death.

Now, you’re probably sitting there, thinking about that splicing thing and feeling vaguely frustrated. “Why do you do that, you stupid cell?” you might be asking. It seems like a lot of effort to spend, doesn’t it? Why go through all the trouble of cutting out middle bits, just so you can string exons together like numbered beads on a string?

Because you don’t have to string together all of them. I mean, you can, and often do, using a process called constitutive splicing. All you have to do is cut out all of the introns, put together all of the exons, and that baby’s ready for translation. But, what if you’re a clever, slightly self-absorbed eukaryotic cell, and you want to prove your ingenuity to the rest of the world? What else could you do with splicing?

Well, you could leave out certain bits. Protein A contains Exon X? Well, if you leave out Exon X, you could make Protein B. Seems like you could only do damage that way, but actually, you can come up with some pretty neat and useful combinations. In fact, this happens a lot, and it means that our cells cleverly engineer several different kinds of proteins from single genes. This means, among other cool things, that a gene expressed in one tissue can produce a different protein than the same one expressed in another tissue.

So, we’ve added stability to our transcript using a 5′-cap and a poly-A tail, and we’ve gotten creative using alternative splicing. Surely our cells are ready to leave these poor things alone…? Surely?

But no, because our cells aren’t content with just snipping out bits of what’s already there. No, they actually go and edit the code of their mRNAs.

You see, when you strip away all of the fancy nomenclature, the bases in RNA are still just chemical structures, and we have plenty of enzymes that can mess with those. We can pretty easily turn an adenine (which bonds to uracil) into an inosine (which bonds to guanine) or a cytosine (which bonds to guanine) into a uracil (which bonds to adenine).

To turn an adenine into an inosine, you need an enzyme called adenosine deaminase, which only works on double-stranded parts of mRNA. To make cytosine into uracil, you need a structure called an editosome, which makes use of cytosine deamination functionality. Either way, changing nucleotides changes their codons, changing the amino acid sequence, introducing new splice sites, or creating new stop codons.

All right, I bet you’ve had enough of eukaryotic shenanigans, haven’t you? I know I have. However, I’ve still got a quarter of a Mountain Dew left, so we’ve gotta keep going. Since we’ve gotten to the end of the material for my test, I’m going to take you back—back to the very beginning, where we talk about the structure of nucleic acids in the first place.

Questions? Comments? Hold that thought, I’ll be back in five—



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