lec 8.2 - translation (initiation and elongation)

initiation in prokaryotes required components

• need both ribosomal subunits, initiator amnioacyl tRNA and mRNA

• needs ribosome binding site (RBS) - shine dalgarno sequence

  • AGGAGGU (upstream of AUG)

  • positions ribosome small subunit at correct position to find the first AUG

• bacteria sometimes has internal mRNA

shine-dalgarno – upstream of AUG by 8-13 nucleotides

• sequence is complementary to rRNA

  1. tRNA w/ fMet at P site

  2. second aa on tRNA at A site

• ribosome can also use internal ribosome entry sites (IRES)

besides the shine dalgarno RBS, what else can ribosomes use?

• ribosomes can also used IRES (internal ribosome entry sites)

• these allow another gene to be translated at once (2 in total)

polycistronic genes in bacteria

• can have overlapping polycistronic genes in bacteria

what do eukaroytes have instead of shine dalgarno?

• describe it

• Kozak sequence

• usually a purine - 3 residues before the start codon and a G immediately following the start codon

• Kozak sequence makes contact with tRNA → enhances translation (not an RBS like Shine-dalgarno)

  • contact w/ initiator tRNA

  • enhances translation

• correct AUG in eukaryotes is AUG closest to 5’ cap (first one after), not like this in prokaryotes

initiation in bacteria

• requirements

• products

• requires 3 initiation factors

  • IF1, IF2, IF3

• produces initiation complex and tRNA carrying formyl-methionine

initiation in eukaryotes

• requirements

• products

• uses 5’ cap to recognize, then scan to find first AUG

• 12 initiation factors

• binding and hydrolysis of GTP and ATP

polysome - in prokaryotes

• what is it?

• what does it do?

• what allows for this phenomenon?

• many ribosomes on a single mRNA (which is still being transcribed)

• increases rate of translation

• can happen because both transcription and translation occur in the cytoplasm (no nucleus so mRNA does not have to travel out of it) and no introns need splicing

where does translation start in viruses

• can start translation at IRES site (internal ribosome entry site) when 5’cap missing – used by viruses and bacteria

• IRES recognized by eIF4F initiation factor

elongation in prokaryotes

• requirements/factors

• 3 elongation factors in bacteria

  • EF-Tu

  • EF-Ts

  • EF-G

• Tu and Ts helps with binding of new tRNA with aa and peptidyl transferase reaction

  • transfer new aa on growing peptide chain

  • if tRNA has wrong anti-codon it gets rejected

translocation in prokaryotes

• EF-G (elongation factor G) drives translocation

  • requires energy → GTP hydrolysis

elongation in eukaryotes

• ribosome removes the exon junction complex (EJC) complex as it proceeds along the mRNA

requirements of termination in prokaryotes

• requires 3 termination or release factors

  • RF1, RF2, RF3

steps of termination in prokaryotes

• happens in 2 steps, needs RF1, RF2 and RF3

• step 1

  • RF1 recognizes stop codons UAA and UAG

  • RF2 recognizes stop codons UAA and UGA

  • they activate ribosome to hydrolyse peptidyl tRNA

• step 2

  • RF3 (GTPase) releases RF1 and RF2, binds GDP → hydrolyzes GTP to signal termination is done

• at the end of this step translation is terminated but ribosome complex is still together therefore need RRF along with EF-G to dissociate complex

ribosome recycling

• uses RRF (ribosome recycling factor) to dissociate ribosome complex by wedging the ribosome apart

• RRF mimics tRNA shape and recognizes stop codon

• use EF-G-GTP and GTP hydrolysis (energy) to move RRF into P site