Translation
tRNA
- amino acids not free in cell, carried by tRNAs
- all are 70-80nt long
- make intramolecular basepairs to form three stem-loops and a stem
- all have a three nucleotide sequence in one stem-loop, that anticodon, that basepairs with mRNA during translation
o codon 5’-3’, anticodon 3’-5’
- all accept the amino acid on the acceptor stem, covalently linked to 3’ A (all have - CCA3’, this is added after transcription)
- many modified bases in tRNAs, modified after txn
- bacterial usually 30-40 different tRNAs
o but 61 codons – this is a problem, some tRNAs must recognize more than one codon
Wobble
- this is how one tRNA can recognize more than one codon
- first two basepairs (5’ in codon, 3’ in anticodon) for standard basepairs
- last one can be nonstandard
o anticodon with G in 5’ position can basepair with codon with C or U in the 3’ position
§ any codons with NNPyr always encode same amino acid and can be recognized by an anticodon with G at 5’ end
- inosine is frequently found at 5’ end of anticodon – can basepair with A,C,U
- show Fig.8.22 for rules
Aminoacyl tRNA synthetases
- there are 20 of these enzymes, link one amino acid to all compatible (cognate) tRNAs for that amino acid
- amino acids ends up linked to 3’OH of terminal A in tRNA
- the synthetases are vital, since ribosome only checks tRNA-mRNA basepairing and doesn’t care what amino acid tRNA is carrying
Ribosome
- the enzyme that reads mRNA sequence, allows tRNAs to bind codons, and links the amino acids on the tRNAs together
- ribosomes are composed of stable rRNAs and many proteins, assembled in a precise way
o large and small subunits of the ribosome
§ large (50S and 60S) has two rRNAs and a number of proteins
§ small (30S and 40S) has one rRNA (16S) and a number of proteins
§ rRNAs are denoted in Svedberg units – this is how fast they sediment in centrifugation (23S and 5S in bacterial large, 16S in bacterial small)
§ large and small subunits and whole ribosome also in S units (bacterial is 50+30S = 70S, eukaryotic 60S+40S = 80S)
Translation
Ribosome “reads” open reading frame and makes protein
- 5’ UTR before start codon
- 3’ UTR after stop codon
Translation initiation
Bacterial
- 30S subunit binds initiator fMet-tRNAMeti
o both prokaryotes and eukaryotes have a special initiator tRNA that accepts Met.
o in bacterial, Met is formylated ( attached to amino group)
- 30S + fMet-tRNA finds start codon
o finds AUG because just upstream of this is a conserved sequence (Shine-Delgarno) that can basepair with the 16S rRNA
o so can find all AUGs on a mRNA because all have Shine-Delgarno sequences by them
- then 50S subunit joins
- fMet-tRNAMeti ends up at tRNA binding site called P site
Eukaryotic
- some similarities, some differences
- 40s subunit + tRNA Meti make a complex
- this whole complex binds at the 5’ cap and seems to move along mRNA until it reaches the first AUG in a good consensus (Kozak) sequence (5’ACCAUGG3’)
o will skip one in bad consensus
- now 60S large subunit comes in
- tRNA Meti ends up at P site
Elongation
1) incoming tRNAs bind at A site
2) large subunit rRNA catalyzes peptide bond formation between amino acids, severing covalent bond between tRNA at P site and amino acid
3) translocation
o tRNA at P site goes to E site
o tRNA that was at A site goes to P site
o new tRNA comes into A site
Termination
- termination factors enter at A site
- everything goes its separate way
o large subunit
o small subunit
o mRNA
o tRNAs
o newly synthesized protein
Polysomes
- many ribosomes can simultaneously translate a single mRNA
- circular polysomes and recycling and translation efficiency
o circular mRNA 5’ and 3’ ends held near each other by protein-protein interactions between proteins that bind cap and bind polyA tail
Processing after translation
- Fig 8.26 shows
o Removing initiator Met usually happens
o Cleaving polyprotein rare
o Modifying protein very common
Problems
21-26 and 28 are thought provoking to me……