Lec 18 - Protein Synthesis & Regulation Flashcards
ribosome
diff subunit function? amount of RNA vs protein?
- large (peptide bond formation) & small subunits (decoding)
- ~2/3 RNA, ~1/3 protein
tRNA & tRNA binding sites
three sites
- tRNA is an adapter b/w nuc sequence and aa; capable of base pairing with mRNA on one end // holds aa on other end
- three tRNA binding sites (APE)
- a = incoming aminoacyl-tRNA enters ribsome
- p = holds growing **peptidyl-tRNA ** chain
- e = where rRNAs exit
R to L
base pairing b/w tRNA and mRNA
wobble base-pairing
inosine wobble pairing? occus at which codon?
occurs in ribosome
- occurs at 3rd codon position only
- G-U wobble pairing can occur with U on tRNA or U on mRNA
- inosine wobble pairing: anticodon may contain inosine (adenosine –> inosine via deamination)
- inosine can pair with C, U, or A (purine, purine)
tRNA
transcribed by what? work with what?
transfer RNA (tRNA)
- tRNA are transcribed by pol III; distinctive conserved structure
- all tRNAs work with ribosome (similar, not identical)
- many chemically modified bases
- gold standard for tertiary stucture in RNA molecules
(anticodon rests at bottom of tRNA & attachment to aa at 3’ end)
tRNA biogenesis & aminoacylation
biogenesis
- endonucleases trim off both ends of rRNA sequence
- separate endonuclease (not spliceosome) removes additional seq from anticodon loop
- at very end of 3’ end of mature tRNA is a CCA seq; this is added post transcriptionally; tRNA cant accept AA until CCA is added
aminoacylation
- tRNA is “charged” with an AA (-COOH) through a high energy ester bond
- this is the thermodynamic drivng force for peptide bond formation
- charging occurs on 3’OH of terminal A residue (of CCA frame)
aminoacyl-tRNA synthetase
aminoacyl-tRNA synthetase
- use ATP hydrolysis to charge them w/high energy ester bond
- AA + tRNA + ATP –> aminoacyl-tRNA + AMP + PPi
- synthestase + tRNA determine genetic code; 1 synthetase per AA (20)
charging of tRNAs
tRNA synthetase mechanism + proofreading
job of tRNA synthetases? nucleophile? 2 synthetase classes?
- tRNA synthetases have important: to charge correct tRNA with correct AA (distinguish AA b/w steric exclusion, size)
- AA’s acid is the nucleophile
- 2 synthetase classes (class 1) 2’-OH is nuc (class 2) 3’-OH is nuc
- final prod always has 3’-O linked to the AA
proofreading
- distinguish b/w valine & isoleucine (1/200 times valine charged instead of isoleucine)
- accuracy increased 3,000 fold by having 2nd active site too small to accomodate isoleucine (hydrolyzes valine if added)
- synthetase must also pick correct tRNA; does recognize anticodon, but also have specific bases outside that can be recognized
translation overview
5 general steps?
1) activation of AA: tRNA is aminoacylated
2) initiation: mRNA and minoacylated tRNA bind to small ribosomal unit; the large subunit then binds
3) elongation: cycles of aminoacyl-tRNA binding & peptide bond formation until AA reaches stop codon
4) termination: stops when stop codon encountered; mRNA & protein dossociate & rib subunits recycled
5) **protein folding ** & posttranslational processing
translation elongation
- charged tRNA recruited to ribosome
- peptide bond formation to extend growing polypeptide chain
- translocation to shift ribosome forward to next codon
EF-Tu (bact) / e-EFIa (euk)
what is it? function? what puts it in A site?
- elongation factor that binds to charged tRNA and brings it to A site of the ribosome
- GTP hydrolysis puts tRNA into A site
- nucleotide exchange (swaps new GTP for GDP) recycles EF-Tu / eEFIa
kinetic proofreading
base paring b/w what inc fidelity? pep bond formation occurs when?
- codon-tRNA basepairing = translation fidelity; promotes GTPase
- delay after GTP hydrolysis promotes dissociation of bad codon-tRNA pairing
- peptide bond formation then occurs with AA in A site attacking AA in P site
also, the ribosome is a ribozyme (RNA enzyme)
inc fidelity just by waiting
EF-G (bact) / eEF2 (euk)
- another elongation factor following peptide bond formation
- tRNA mimic; interacts with ribosome in similar place
(EF-G and release factors both mimic tRNAs)
subunit rotation
- translocation occurs through rotation of subunits
- EF-G (eEF2 in euk) comes and shoves in (sim to EF-Tu/eEFIa & tRNA), then uses GTP hydrolysis (not to push in), but to reposition ribosome
translation termination; release factor
- no tRNAs recognize stop codons
- release factors are proteins that mimic tRNAs; termination releases peptide from tRNA
- release factors are similat to EF-Tu bound to a charged tRNA
bacterial vs eukaryotic ribosome release
- if RNA damaged/contains frameshift, possible for ribosome to reach 3’ end of mRNA without running across stop codon– causing ribosome to stall
bacterial
- specialized **tmRNA **(transfer-messenger) with 5’ end mimics tRNA and 3’ end that acts as continuation of mRNA
- tmRNA slides into A site & growing peptide chain transferred ont it
- this transpeptidation adds Ala to growing chain
- translocation rescues translation: the mRNA portion of tmRNA now accessible in A site
- translation continues until seq from tmRNA incorporated onto end of stalled polypeptide product; this seq tarfets newly synthesized peptide for degradation
- overall mech rescues ribosome to move on to another transcript
eukaryotes (simpler)
- normally have release factor
- dedicated factors recgnize stalled ribosoe & force it apart
- peptide chain still attached to tRNA & this is also a signal for degradation
