Ribosomes and translation

Question 1

Ribosomes consist two RNP subunits

Answer 1

Ribosomes are larger abundant RNP particles
Have two unequal subunits
Structure is highly conserved
Codon/anticodon binding at small subunit
Peptide bond forming at large subunit
3 non overlapping tRNA binding sites (A P E) found at subunit interface
2 tRNAs bound at any one time (AP or PE)
Peptidyl transferase centre (PTC) on large subunit is where peptide bond occur
PTC is RNA-rich so peptide bonds form because they are RNA-catalysed reaction
A site where charged tRNA binds
P site where tRNA attached to polypeptide chain
E site where non-charged tRNA binds before leaving ribosome complex

Question 2

Ribosome synthesis in eukaryotes

Answer 2

Is complex, energetically expensive, requires hundreds different proteins and RNAS
rRNA transcription and early rRNA processing occur in nucleoli
Later processes occur in nucleoplasm and cytoplasm
Functionally active ribosomes generated after exported from nucleus so protein synthesis only occur in cytoplasm and stops accidental translation of nuclear DNA

Question 3

Essence of translation - peptidyl transfer

Answer 3

Anticodon and aminoacyl group on adjacent tRNA molecules close together on ribosome
Aminoacyl and peptidyl group attached to tRNA by carboxylic ester links
Peptide bond formation (peotidyl transfer reaction) involves alpha-amino group of aminoacyl-tRNA and carboxyl group of peptidyl-tRNA

Question 4

Translation elongation cycle

Answer 4

2 tRNAs bound any time in pre-translocation state (A and P site) or post-translocation state (P and E site)
Translation involves reiterative cycle aminoacyl-tRNA binding, peptide formatioin and translocation of ribosome along mRNA then ejection of non-charged tRNA at end of chain

Question 5

GTPases in translation elongation

Answer 5

Aminoacyl-tRNA brought to ribosome by elongation factor EF1A (EF-Tu in pro)
Charged tRNA don’t exist on own
Translocation required another elongation facotr EF2 (EFG in pro)
EFA1 and EF2 are GTPases
2 GTP molecules hydrolysed only occurs per incorporated correct amino acid
- First GTP spent to ensure correct tRNA binds to A site
- Second GTP spent making chain move along

Question 6

Specific initiator tRNA binds to start codon

Answer 6

EF1A bring tRNA to A site, but start codon tRNA brought to P site, rest tRNA directed to A site
Cell contain 2 distinct methionyl-tRNAs
Elongator methionyl-tRNAs bind to internal AUG codon and associated with EF1A (EF-Tu)
Distinct initiator methionyl-tRNA recognises AUG start codon and associated with elF2

Question 7

16s/mRNA base-pairing selects initiation codon in prokaryotic cells

Answer 7

mRNA sequence longer than sequence that will get translated due to open reading frame
Shine-Dalgarno (SD) sequence within prokaryotic mRNA recognised by bpairing with nucleotides at 3’ end of 16S rRNA
Polycistronic mRNA in prokaryotic cells

Question 8

Translation initiation in eukaryotes

Answer 8

Monocistronic mRNA in eukaryotes
Initiator tRNA^(Met) (bound to elF2) along with small subunit, assembled at 5’ end mRNA through interaction with cap-binding complex (CBC)
Preinitiation complex scans along mRNA, using helicase activity of CBC, until finds AUG codon within appropriate context - the Kozak sequence
After selection start codon, large subunit recruited

Question 9

Translation termination

Answer 9

Stop codons recognised by protein termination (or release) factor, not recognised by tRNAs
Prokaryotes have 2 termination factors (RF1 and RF2) eukaryotic cells have 1 (eRF1)
2 release factors act together to cause termination
Initial binding of RF1 or RF2 (eRF1 in Eu) triggers peptide hydrolysis
Subsequently RF3 (eRF3 in Eu) allows release RF1/RF2 from ribosomes