Lecture 24 Flashcards
(19 cards)
fMet and initiator tRNA
First a.a is modified version of methionine, N-formylmethionine (fMet) in bacteria. Initiator tRNA recognises AUG start codon and brings fMet. Initiator tRNA is different from other methionine tRNAs. Only the start codon, any other AUG brings methionine. fMet is removed from newly synthesised pp chain at ~10a.a production.
Formylation of methionine steps
- Methionine is linked to the initiator tRNA and the normal Met tRNA by the same aminoacyl-tRNA synthetase
- Transformylase formylates the methionine attached to the initiator tRNA. Enzyme cannot formylate free methionine or methionine attached to normal Met tRNA.
Steps for prokaryote initiation
- Initiator factors IF1 and IF3 bind to small 30S ribosome subunit, separating it from 50S subunit to stop premature binding to 70S ribosomal subunit.
- IF2 binds to GTP conformational change in IF2, allowing it to recognise fMet and initiator tRNA complex specifically.
- IF2 binds to fMet-tRNA complex, joining 30S, IF1 and IF3 to form a 30S initiator complex.
- In 30S initiator complex, mRNA bound via Shine-Delgarno sequence to the 16S rRNA and initiator tRNA-fMet complex binds to start codon in the small subunit’s P site.
- Once interactions occur, IF1 and IF3 are ejected from initiator complex while IF2 works to recruit 50S ribosomal subunits.
- Once 50S ribosomal subunit binds, IF2 is removed. Complete 70S initiation complex made. Rate limiting step.
- Now progresses to elongation.
Establishing reading frame and a.a activation
Binding of first tRNA to start codon establishes reading frame. Next tRNA enters through A site by elongation factor Tu (EF-Tu). EF-Tu binds to aminoacyl-tRNA to protect ester linkage between a.a and tRNA (otherwise a.a won’t activate so no peptide bond formation between other a.a) and it checks the accuracy of anti-codon-codon binding.
Elongation differences between initiator tRNA and the other tRNAs
The initiator tRNA is the only tRNA that doesn’t enter through the A site, it starts in the P site. Once both P and A sites are occupied, the fMet in the P site forms a peptide bond with the amino acid in the A site. Peptide bond formation is catalysed by rRNA in ribosome.
Formation of first peptide bond
The ribosome orients tRNAs so that the amino group of the A-site amino acid can attack the ester bond linking fMet to its tRNA in the P site. This forms a peptide bond. After the reaction, the P-site tRNA is left empty, and the A-site tRNA holds the dipeptide.
Elongation steps
- The empty tRNA moves from the P-site to the E-site
- The peptide tRNA moves from A-site to P-site
- Next codon positioned into the A-site.
- tRNA in E-site released by the ribosome. The P-site is filled. A site is empty. Continues until stop codon reached.
Start point of elongation
Small and large ribosomal subunit is bound, mRNA bound, initiator tRNA bound to P site and start codon. A and E sites are empty.
Elongation-translocation
peptide chain attached to tRNA on A-site. Ribosomal subunit rotates causing the peptide chain on A-site is transferred to P-site on large ribosomal subunit. E-site and P-site now occupied while A-site is empty, but on small subunit, positions have not changed. Next codon is still not available for 3rd tRNA to come in. Protein synthesis stops unless everything is moved in the small subunit by one codon. EF-G helps move tRNA and mRNA in SS by 1 codon. Proteins are made in the N to C terminal direction.
Termination
UAA, UAG, UGA. There are no tRNAs that can recognise these codons. They are recognised by release factors. After translocation, the pp is attached to the tRNA in the P-site, leaving the A-site empty. As the next codon has no corresponding tRNA, RF1 comes and binds to the stop codon. It is so big, it traverses the A-side and binds to the P-site. It contacts ester linkage between pp chain and tRNA and hydrolyses it. The p.p chain is released from the ribosomal complex dissociates. They are free to take part in another round of translation.
Difference between euk and prok ribosomes
Euk are larger (increase proteins and rRNA)
Difference between euk and prok initiator tRNA
In euk, it has distinct initiator tRNA but, it brings normal methionine NOT fMet.
Difference between euk and prok initiation and met
Euk translation is decoupled from transcription. No Shine-Delgarno sequence in euk mRNA for initiation. Instead, first AUG is the state site for translation as mRNA codes for one protein product.
Eukaryotic translation initiaton steps
- The 40S ribosome, initiation factors and initiator tRNA form a complex and bind to 5’ cap of mRNA (prokaryotes doen’t have 5’ cap)
- Complex searches for AUG codon, using helicase (powered by ATP) to drive the complex along mRNA strand (5’-3’, 1 position at a time)
- Once AUG is recognised, initiator tRNA binds to it and IF recruit large 60S ribosomal subunit to form full initiation complex. IFs are removed.
- Elongation begins.
Eukaryotic vs prokaryotic mRNA
Eukaryotic mRNA forms a circular structure during translation through interactions between the 5’ cap and 3’ poly(A) tail, which increases translation efficiency and prevents faulty translation. Histone mRNAs lack a poly(A) tail but have a 3’ stem-loop structure that performs a similar role. Prokaryotic mRNA lacks both a cap and a tail, is not circular, and can be translated while still being made. It is often polycistronic, unlike eukaryotic mRNA which is usually monocistronic.
Eukaryotic vs prokaryotic elongation and termination
Specific factors differ. EFs and ribosomal release factors exist in both, but their name and sequence differ.
Eukaryotic vs prokaryotic organisation
Eukaryotic translation machinery is attached to cytoskeleton taught to increase efficiency. Means that mechanoreceptors can increase p.p synthesis.
Inhibiting protein synthesis for drugs
Differences in bacterial and eukaryotic pp synthesis can be used to develop universal (inhibits both) or specific drugs (bacteria only)
C. Diptheriae toxins
Causes diphtheria by stopping pp synthesis in cells. Diphtheria toxin binds to receptors on cells where they are endocytosed. The toxin is cleaved and a fragment targets EF2 by adding ADP-ribose (large) to diphthamide which is an important a.a in EF2. H blocks EF2 function=no translation = no pp synthesis.
