Explain the 2 main events in protein synthesis initiation
1) tRNAmet interacts with EIF2 (in GTP bound state), complex interact with small ribosomal subunit and other pre-initiation factors to ensure it stays separate from large subunit - this creates 43S complex
2) mRNA activation - 5’ end cap is recognized, eIF4 interacts with 43S pre-initiation complexthrough bridging of eIF4G. This brings down small subunit to 5’ end of mRNA. eIF4G has associaiton with PAPBC, forming a loop
Describe GTP hydrolyzation when scanning complex hits AUG
When scanning complex hits AUG, eIF2-GTP is hydrolyzed to eIF2 GDP
Large subunit interacts with small one, creating 80S, which can being elongation
What is first step of elongation cycle?
- When amino aicd charged tRNA enters the Asite
- EFIalpha, GTP and tRNAaa that matches next mRNA codon is allowed to enter the ribosomal A site
Explain conformational changes of the ribosome during elongation
When A site is bound, EFI alpha is released, and GTP hydrolysis causes a conformational change in the ribosome
- Conformation change brings P and A site closer in proximity
What is a ribozyme? What is a vital one in elongation?
A ribozyme is an enzymatically active RNA molecule
Polypeptidyl transferase is responsible for peptide bond formation
- In bacteria, 23S rRNA catalyzes peptide bond formation
- In eukaryotes, 28S rRNA, also doesn’t require protein
What is the “second” hydrolysis in elongation?
GTP is hydrolyzed again, bringing ribosome back to its original conformation.
This causes a forward translocation by one codon, moving tRNAs into E and P sites, which is monitored by EF2-GTP
tRNA in E site is forced out during the next conformational change
What are the steps in translation termination?
- Ribosome encounters STOP codon in A site, which is recognized by eRF1, which binds to site. BVecause there is no remaining amino acid present, chain will be cleaved.
- eRF3 releases the polypeptide chain, hydrolyzes GTP to cleave last amino acid
- peptide cleavage releases the polypeptide chain and tRNA present at the E site
- The complex breaks apart, leaving a large and small subunit
How can we force mRNA to favour reinitiation?
- proteins associated with the 3’ end of the mRNA interact with proteins at the 5’ end
- These loops favour re-initiation of translation, and reduce the ability of nucleases to “chew up” mRNA
- Highly translated mRNAs are also highly stabilized
Explain polysome profiles?
Put over a centrifugal gradient - can identify the types of ribosomal containing different things;
- 80S means initiation
- many small humps increasing in molecular weight correspond to mRNAs with more ribosomes associated with them (polyribosome)
