Lecture 23 (RR11): Translation I Flashcards
What are the 3 roles of RNA in protein synthesis
All three classes of RNAs are required to synthesize a polypeptide. We can see this in the example below.
mRNA has all the instructions encoded into the triplets that are being read by a ribosome that is made of proteins and rRNA in addition to these **tRNAs **that come in and bring the amino acids into the ribosome as it translates the mRNA to make a protein.
rRNA
- rRNA accounts for approximately 80% of the total cellular RNA. Therefore, rRNA makes up the bulk of RNA within the cell.
- Critical for carrying out so many of the major processes involved in translation.
- It is transcripted from these rDNA loci on the chromosomes in specific regions and having the transcription of these clusters is sufficient to set up membraneless liquid-liquid condensates that we call a nucleolus. A nucleolus is an rRNA factory - critical for forming all the ribosomes and assembling some of these structures so that they can be exported into cytoplasm where they will be involved in protein translation.
Ribosomes
rRNAs contribute to the formation of the ribosome by associating with given proteins to give rise to the large subunit of the ribosome.
In the picture, we can see the large subunit and small subunits that come together to make the ribosomes. S is for Svedberg units and these are not additive, so when they join together they have a different confirmation and so they sediment differently in the centrifugal gradient.
Eukaryotic organisms are very similar in their composition. They require the equivalent RNAs with one additional RNA in order to give rise to the large and small subunit. It is a combination of RNAs and proteins that give rise to these protein translation factories.
The complex secondary structure of ribosomal RNA is highly conserved among the thousands of species investigated to date.
- This is a 16S bacterial RNA and it is similar to 18S eukaryotic rRNA.
- Complexity with which it folds up into stem loops and hairpins is very conserved depending on the sequence. The way it folds up seems to be conserved by prokaryotes and eukaryotes.
- This conservation suggests that
→ Prokaryotes got it back in the primordial world by generating this structure that is critical for forming ribosomes and must carry out an essential role in that process.
→ Structurally many of these stem loops are not random. They are carrying out critical roles within the ribosome.
→ You get a different structure with every ribosomal RNA. They fold up into very distinct structures.
Pre-tRNAs undergo critical processing
tRNAs are class III gene products that have this important structure that has to be modified in order for them to actually be mature tRNAs that can be used in the translation reaction.
Pre-tRNAs are transcribed by RNA Pol III and thereafter are processed to yield mature tRNA:
1) the 5’ end sequence is removed
2) a short segment is removed
3) CCA is added on to the 3’ ends (this is the site where the tRNA will be bound to its cognate amino acid through an ester linkage → this is why all tRNAs have the CCA, the adenosine sets up the ester bond, 3’ or 2’ hydroxyl linkage.).
4) extensive modification of internal bases
The binding of the cognate amino acid to the tRNA
1) Aminoacyl tRNA Synthetases recognize and bind to their cognate tRNAs. Aminoacyl tRNAs Synthetases are enzymes necessary to link the amino acid. There are 20 of these enzymes and they recognize each amino acid that is present in our cells. They will carry out the catalytic reaction and link the amino acid to the tRNA at that specific site, 2’ hydroxyl or 3’ hydroxyl on the adenosine terminal. The formation of the ester bond is critical and requires ATP.
2) Once the tRNA is bound to the amino acid, it is considered to be charged. Ready to interact with the appropriate site in the ribosome.
**Important: **
- A single aminoacyl tRNA synthetase can bind to more than one unique tRNA.
- A single tRNA can bind to more than one single codon → in the context of the ribosome, it allows for non Watson Crick base pairing to occur. This is one explanation for the wobble that we see between codon and anticodon.
- The code is therefore considered “degenerate” → it is no 1 to 1 to 1.
The tRNAiMet
The tRNAiMet is needed to start synthesis of a polypeptide chain
Two types of tRNAs exist for the methionine codon
* Most proteins begin with methionine amino acid because two different types of tRNAs encode methionine.
* Both are charged by the same aminoacyl tRNA synthetas
* Initiator tRNA met has to be distinct from regular tRNA met:
→ tRNAMet is exclusively used for elongation of a polypeptide chain. Regular tRNA met would encode methionine in the body of the peptide chain (somewhere in the middle).
→ tRNAiMet is exclusively used for initiation of a polypeptide chain → only at first position, it is structurally different then the one used for elongation. It can interact specifically with the P-site of the ribosome.
* The methionine in bacterial tRNA i is modified by addition of a formyl group that distinguishes it from the tRNA met that would be utilized in an elongation of the polypeptide chain.
* This is all due to the structural characteristics of the initiator tRNA met that allows it to interact with the P-site (peptidyl site) on the small ribosomal subunit.
* All the other tRNA met interact with the A site (the aminoacyl site).
Assembly of the pre-initiation complex
- Initiator of tRNA met interacts with an interaction factor. For the initiation of protein synthesis there is a whole set of critical proteins that work together and are essential for the initiation of most proteins.
→ In eukaryotes we refer to these as eIF - When protein synthesis is not ongoing, it is important to keep the ribosome subunits separate from one another. This is achieved by the interaction of specific initiation factors (eIF1, 1A and eIF3) with the small ribosomal subunit. In this conformation, the small ribosomal subunit can’t interact with the large ribosomal subunit and can’t give rise to a competent ribosome.
1) When protein synthesis is favored or induced, the initiator tRNA met will interact with one of these initiation factors called eIF2 which is a GTP binding protein.
2) When eIF2 is bound to GTP, it can interact with the initiator tRNA met and will form a tertiary complex.
3) That complex can interact with the small ribosomal subunit to give rise to the 43S preinitiation complex which contains all the eIFs, the small ribosomal subunit and the initiator tRNA met with eIF2 in its GTP bound state.
This process is highly controlled → if growing conditions are not optimal, very often eIF2 can be modified by phosphorylation and that phosphorylation will block its ability to interact with initiator tRNA met usually by blocking its ability to bind GTP.
- Protein synthesis can be negatively regulated by phosphorylation of eIF2. By phosphorylating eIF2 at the correct place, you can block protein synthesis.
- When you are starving or under conditions that are not great and you want to block protein synthesis because it takes a lot of energy, you can impinge on this reaction by phosphorylating eIF2 so that it can no longer interact with the initiator tRNA met and so you will never form a 43S preinitiation complex.
5’ Cap
- The mRNA has this 7-methylguanylate structure at the 5’ end that is bound by a Cap binding protein during cytoplasmic remodeling and that cap binding protein is critical because it blocks the 5’ end so that ribonucleases cannot chew it up. It is also very important for recruiting in critical factors involved in protein synthesis.
- Because the dimeric guanylyltransferase binds to the phosphorylated C-terminal domain (CTD) of RNA polymerase II only mRNA transcripts are capped!
- Therefore, only class II (mRNA) transcripts are efficiently translated!
A cap-binding protein is required for efficient translational initiation
* Sonenberg et al. purified a protein that bound the 7mGDP cap. A protein that interacts specifically with the 7mGDP cap.
* Addition of the purified protein to capped mRNAs increased the translational efficiency in vitro, but did not affect uncapped transcripts.
* When he used cap mRNAs, he could increase protein synthesis of those templated by adding in extra protein (the protein that he identified that interacted with the cap).
* This suggests that translation is limiting for this protein, if you add more it increases the efficiency of translation. This is cap dependent.
Role of eIF4
eIF4 is instrumental in recruiting an mRNA to the preinitiation complex
* They identified a complex that was critical for recognizing the 5’ end of an mRNA to be translated and this complex was eIF4.
* Within the eIF4 was the cap binding protein. The cap binding protein is eIF4E.
* eIF4E and its associated subunits will interact with the 5’ end of an mRNA that is going to be translated in a cap binding protein dependent manner. eIF4E will recognize the 7- methylguanylate structure and I will interact strongly with it bringing all of these other subunits to the 5’ end of the mRNA. This step is referred to as the mRNA activation.
* Activity of the cap-binding subunit eIF4E is under tight regulation; overexpression of this protein is associated with tumour formation.
* eIF4G, which is shown interacting directly with the cap binding protein, plays an important role. It can interact with one of the initiation factors present on this pre-initiation complex eIF3. It can bring in the small ribosomal subunit with all the initiation factors (including the initiator tRNA met) to the 5’ end of the mRNA to be translated.
* eIF4G interacts with the Poly A binding protein, interacting with the poly A tail. So, eIF4G associates with PABPC (cytoplasmic version of poly A binding protein) and it pulls the poly A tail up into the complex to form a loop.
* So, eIF4 brings in the small ribosomal subunit and the pulls up the tail via its interaction with poly A binding protein to make weird loops
Interactions between the 5’and 3’ end of the translated mRNA
Interactions between the 5’and 3’ end of the translated mRNA favour reinitiation
* You can see the loops in vitro by doing electron microscopy.
* Proteins associated with the 3’ end of the mRNA interact with proteins at the 5’ end.
* These loops may favour the re-initiation of translation. You could initiate translation, the ribosomes could read all the way through the mRNA and finish off exactly where they started, where they can re-initiate again and again. These loops make translation efficient because you keep all the proteins together around the region where initiation will occur.
* Efficient translation often is associated with mRNA stabilisation
* All of the EIF4 proteins are important for generating a structure that is critical for efficient translation (these loops), which are conferred by a protein protein interaction between eIF4G and PolyA binding protein present in the cytoplasm (PABPC). This puts the 3’ end of the mRNA close to the 5’ end .
eIF4 components
These two eIF4 components are needed for initiation:
* eIF4E-Cap
* eIF4G-binds eIF3 and PABPC
- eIF4A-RNA helicase (ATP) → ATP dependent RNA helicase (remove secondary structure/ melt double stranded RNA.
- eIF4B-enhances eIF4A activity → it appears to enhance the activity of its twin brother: eIF4A (it was thought to be a scaffold protein at first).
Process continued (what happens):
1) eIF4A - associated RNA helicase activity removes any secondary structure in the 5’ region of the transcript.
2) What happens to eIF4E?
New information suggests that eIF4E remains associated with the scanning complex forming a loop at the 5’ end.
3) This complex will scan through the 5’ region of the mRNA to be translated, removing any secondary structure that might be present there and the scanning will continue in an ATP dependent manner because it is driven by eIF4A (RNA helicase).
4) Don’t think of it as a straight line but instead a loop
As the scanning complex moves along the small ribosomal subunit will find itself centered over an AUG start codon.
5) At that point the initiator tRNA met will recognize that codon and will then induce eIF2 to hydrolyze the GTP that it is bound to to GDP. Associated with conformational change.
6) 48S initiation complex - the small ribosomal subunit, all of its initiation factors along with eIF2 that is now GDP bound state and the initiator tRNA met bound to the start codon (AUG) at the p-site.
- when the scanning complex associates with the AUG start codon eIF2-GTP is hydrolysed to eIF2-GDP
7) It is only at this point that the large ribosomal subunit can actually join the small ribosomal subunit and in a reaction that happens instantaneously all of those initiation factors will leave once the large subunit joins the complex.
**Key things: **
- There is a conformational change associated with recognizing the AUG
- The conformational change is mediated by eIF2 GTP hydrolysis to GDP
- Large subunit will join while other initiation factors leave.
This is when you actually generate a competent ribosome.
Functioning steps of a competent ribosome
Stable binding of the initiation complex with the start codon triggers subunit joining and formation of the 80S ribosome.
* the large subunit interacts with the small subunit. It is bound to mRNA substrate very tightly to the extent that it won’t release until it gets to a stop codon.
* The initiator tRNA met is positioned over the AUG, start codon, which is centered over the p-Site.
- Centering it over the p-site ensures that the A site is vacant and ready to accept any charged tRNAs that might satisfy the codon requirement present.
- THis is the 80s initiation complex and it is competent to carry out protein synthesis. It is the protein synthesis machine.
- GTP hydrolysis steps ensure that several contingencies are satisfied before translation begins
- Once translation is initiated the ribosomal complex becomes irreversibly bound to the mRNA until translation is terminated. Won’t let go until it hits a stop codon.
