Lecture 24 (RR12) Translation II Flashcards
Elongation
(step 1 and 2)
Entry of the next amino acid-charged tRNA into the A site starts the elongation cycle.
1) Elongation is entirely dependent on charged tRNAs associated with specific elongation factors:
→ The A site remains vacant due to the interaction between the initiator tRNA met and the P site in the ribosome.
→ Because the A site is vacant, it allows a complex of the elongation factor, EF1α*GTP (a EF1α in a GTP bound state) and a tRNAaa is allowed to enter the ribosomal A site.
→Then, the anticodon-codon reaction will be assessed (see if anticodon matches the next mRNA codon) and if that interaction isn’t acceptable, the tRNA will leave the a-site and allow for another charged tRNA to come into the A site and try again.
→ When the appropriate tRNA charged with its correct amino acid enters into the A site and is capable of interacting with the codon then the GTP in the EF1α will be hydrolyzed and in doing so it will change the confirmation of the ribosome.
2) GTP hydrolysis promotes a conformational change in the ribosome
→ Upon binding of the complex in the A site, EF1 alpha is released and concurrent GTP hydrolysis leads to a conformational change in the ribosome.
→ This change brings the amino acids linked to the tRNAs in the P and A site into close proximity - they come into close proximity with each other.
Why do the amino acids linked to the tRNA in the p-site and the a-site come into close proximity?
In this image, we can see that the amine from the charged tRNA in the A-site comes into close proximity with the carboxyl group from the charged tRNA from the P-site. This will give rise to a peptide bond (does not happen on its own, it requires some sort of enzyme).
What is so special about the ribosome in terms of reactions?
They were able to show that RNA could catalyze the peptide bond all by itself.
* The ribosome contains a “ribozyme” ribozyme -> an enzymatically active RNA molecule.
* The peptidyltransferase reaction is mediated by an RNA in the 23S or 28S.
→ the 23S rRNA (bacteria) catalyzes the peptide bond without any protein requirement, the same applies to the 28S rRNA in eukaryotic ribosomes.
This example along with the fact that RNAs can splice themselves in self splicing reactions, tell us that RNAs might have been the first macromolecules that were present in the primordial earth. In a very early time on earth, perhaps it was RNA that was initiating all the very early catalysis and eventually DNA and protein came into play.
The ribosome now contains a “ribozyme” —> it is considered a ribozyme because it carries out the formation of a peptide bond between amino acid one and amino acid two within the ribosome.
Elongation steps 3 and 4
Following the formation of that peptide bond between those two amino acids, the growing peptide chain finds itself associated with the tRNA that is present at the aminoacyl site (A site).
* In order to go through another phase of adding an amino acid peptide chain, the ribosome has to move forward to vacate this A site so another tRNA that is charged with its cognate amino acid can come in.
* Upon peptide bond formation, a conformational change in the ribosome results in its forward translocation by the distance of one codon-thereby moving the tRNA molecules into the E and P site respectively. This is monitored by EF2-GTP (a GTP binding protein). EF2 will hydrolyze GTP to GDP, in doing this it ensures that the ribosome only goes forward 3 nucleotides.
* A new elongation cycle can begin (the A site is vacant and the growing polypeptide chain is now associated with the tRNA that is present at the P site (peptidyl site).
* E Site = exit site
In the next elongation cycle…
- The tRNA in the E site, now rid of its amino acid, is forced out of the ribosome during the conformational change associated with EF1α release and GTP hydrolysis.
- The growing polypeptide chain is always covalently linked to one of the tRNAs in the P or A site
- The conformational change in the ribosome forces the amino acids that are attached to the tRNA at the A site to be in close proximity to the growing polypeptide chain, particularly the amino acid that is at the very closest region. The 23S or the 28S RNA will catalyze this reaction. Once again, the polypeptide chain will be linked to the tRNA that is in the A site.
- The conformational change kicks out the tRNA that was present in the Esite so that it can be recharged and reutilized in reaction.
- Cycle repeats. Elongation cycles are controlled and regulated by GTP hydrolysis.
Translation termination
Translation termination involves termination factors that recognize the stop codons
* Once the ribosome encounters a STOP codon in the A site, this codon is recognized by a termination (release) factor, eRF1.
* eRF1 is thought to structurally mimic aminoacyl-tRNAs. It is not a tRNA but its structure folds up to look like a tRNA. It fits into the pocket of the A site. The ribosome tries to make this link between the amino acid that would normally be present on a charged tRNA but it doesn’t do it. The result is the polypeptide present at the p-site will be cleaved. This is all dependent on GTP hydrolysis associated with eRF3.
* GTP-bound eRF3 that is associated with eRF1 bound to the A site undergoes GTP hydrolysis
What terminates translation?
eRF3 terminates translation by releasing the polypeptide chain
* hydrolysis of the GTP bound by eRF3 is necessary for the cleavage of the last amino acid in the P site from its tRNA.
* peptide cleavage precedes the release of the polypeptide chain and tRNA present at the E site
* The post-termination complex is finally actively dissociated to yield a large and small subunit ready to initiate another translation cycle. Complete dissociation of the ribosome, the large subunit will leave, the small subunit will leave associated with its initiation factors to ensure that the large and small subunit don’t come together and any tRNAs that are present will also leave
* MRNA can then be translated once again by reinitiation of those same components.
* Theoretically, we have loops so that when these components of the protein synthesis reaction dissociate, they simply come back together at the 5’ end of the mRNA. It is very rare that you will ever see an mRNA with one single ribosome transitioning through it. A mRNA that is heavily translated will not have one ribosome, will have many transitioning at the same time.
Stability of mRNAs
- MRNAs that are expressed in high levels, so making lots of protein, these tend to be stabilized. They are more stable then mRNAs that are weekly translated.
- Usually, mRNAs that are highly translated have longer polyA tails. By having longer polyA tails there is more Poly binding protein, this adds to the protective properties of these proteins. In addition, they will have a greater ability to interact with eIF4 at the 5’ end of the mRNA.
- By forming these circles all of these proteins together tend to protect presumably both the 5’ and 3’ end of the mRNA.
- The length of poly A tail contributes to stability and the binding of the proteins to that poly A tail also contributes to stabilizing these mRNAs.
Polysome profiles
Polysome profiles can be used to quantify translational activity/efficiency.
* Centrifugation can be used to resolve complexes isolated at various stages of translational engagement.
* We can isolate all of these complexes by taking a complex sample (cytoplasmic extract or wholesale extract) and putting that on a cushion of sucrose (gradient of sucrose). By running those proteins that are present at the complex sample in a centrifugal gradient, you can end up separating all of these different complexes based on components that make them up.
* We see the 40s hump that correspond to the small subunit, it finds itself at the top of the gradient because it is pretty light, it does not sediment as fast as the 60S (large subunit)
* The 80s, the initiation complex, is a ribosome bound to an mRNA. In this sample there is a lot of it at the point of initiation.
* All the little humps correspond to mRNAs with multiple ribosomes, called polyribosomes.
* We can examine translation and where translation is being regulated for any transcript by carrying out these kinds of simple centrifugal experiments to separate all of the different complexes out.
