Prokaryotic translation Flashcards
ribosome structure
70s in total
with large 50S subunit: contains 23S + 5S + 31 proteins
and small 30s subunit: 16S + 21 proteins
peptidyl transferase reaction in 50S ribosome subunit (ribozyme activity)
in 23S rRNA
N3 of nucleotide A2486 (of ribosome) accepts proton from amino group of aa-tRNA in A site
this enhances -ve charge of the amino group so attacks the bond between the peptide and tRNA in the P site
N3 of A2486 H-bonds to oxyanion in tetrahedral intermediate and stabilises and accelerates reactions
3’-OH of tRNA in P site accepts proton from A2486
where is the polypeptide exit tunnel in ribosome?
through pore in centre of large 50S subunit and is wide enough so protein can start folding and it protects it from degradation
isoaccepting tRNA
multiple codons so multiple tRNA for same AA
aminoacyl-tRNA synthetases
charge tRNAs with AAs and have specificity
rare to get wrong AA on tRNA
cloverleaf model of tRNA
D loop: 12 unpaired bases with 2-3 dihydrouracil residues (nucleoside)
anticodon loop: 7 unpaired bases w/ anticodon matching mRNA
5’ U and 3’ alkylated purine
variable loop: varies in size, recognise right AA and interact with enzymes
T loop (TΨC loop): 7 unpaired bases, TΨCG binds A site in ribosome
3’ end CCA recognise and bind AA
stems in between loops have closely controlled sizes and give tRNA structure
tertiary structure of tRNA phe in yeast
form L shape with D and T loop forming corner of L
most bases stacked to stabilise apart from 3 anticodon and CCA-3’ so can interact with mRNA or aa-tRNA
effects of antibiotics like erythromycin/chloramphenicol
bind peptidyl transferase centre of 23S rRNA so easily get antibiotic resistance is make single base changes
Colicin E3 protein
inhibits bacterial cell growth that lack Col plasmid
cleaves 16S rRNA 50 nucleotides from 3’ end so mRNA can’t bind to ribosome
streptomycin
binds 16S rRNA of 30S subunit
interferes with fmet binding so prevents initiation of protein synthesis in bacteria
RBS
ribosome binding site upstream of start codon
Shine-Dalgarno sequence
purine-rich region complementary to initiator sites of mRNA
what happens if ribosome is washed in high salt?
30S subunit can’t initiate protein synthesis
initiation requires free 30S
initiation factors and their function in initiation of prokaryotic translation
IF-3 is 22kDa and binds the 30S subunits preventing it from associating with the 50S subunit so can’t activate whole ribosome (prevents premature association)
IF-1 is 9kDa and binds near A site so directs fmet-tRNA to P site (modifies A so only go to P, first tRNA starts in P not A like usual) (prevents premature binding to A site)
IF-2 is 120kDa and reacts with fmet-tRNA and GTP to form complex and delivers complex + mRNA to partial P site in 30S
when 50S joins it triggers GTP hydrolysis and starts process
once the complete 70S complex is formed, the IFs are released and GTP hydrolysed
elongation definition
codon-directed binding of incoming aminoacyl-tRNA, peptide bond forms and ribosome translocated 5’-3’ along mRNA
explain the process of elongation in prokaryotic translation
EF-Tu helps bring and join aa-tRNA to the ribosome but can’t attach until proofread
binding requires EF-T (EF-Tu and EF-Ts) and GTP
(EF-Tu doesn’t react with fmet-tRNA which is why it is not delivered to the A site)
after proofreading, the peptide bond can be formed with help of peptidyl transferase, the aa part of fMet-tRNA is transferred to new aa of 2nd tRNA in the A site
ribozyme function of 23S catalyses this w/o energy
the high energy bond between aa and tRNA is protected by elongation factors
translocation of ribosome along mRNA 5’ to 3’ by 1 codon by EF-G
N-terminal of EF-G mimics tRNA so goes in A site to push ribosome along
EF-G/GTP binds, tRNA like domain interacts with 30S subunit close to partial A site
GTP hydrolysis causes conformational change in EF-G so arm goes deeper into 30S and forces peptidyl tRNA from A to P site
