Translation

1
Translation
mRNA directs synthesis of a polypeptide Action
happens in cytoplasm and on polysomes (ribosomes
mRNA) Simply mixing mRNA with ribosomes
negative tln.many other things involved.
Complex problem how solved?
2
How was tln figured out? Early fractionation of
macromolecules based upon the SIZE and SHAPE of
the structures in velocity sedimentation
gradients (or SUCROSE gradient centrifugation).
Large stuff goes to bottom
S Svedbergs measurement of size of the
macromolecule
Resolving power of sucrose ID RNAs involved.
3
Genetic Code how bases are read in TLN process.
Each word in the code is based upon a stretch
of 3 basesthis is a codon (or triplet). The
genetic code is NON-OVERLAPPINGearly on this was
thought to be so. WHY? Because a single base
mutation would give only a single AA change with
this modelwith the overlapping code, point
mutations would alter three amino acids
Note Shifted reading frames are possible! Cell
could utilize gt1 reading frame to double or
triple coding potential.increasing genetic
capacity at each locus.
4
Number of letters in the genetic code with 20
naturally occurring amino acids 43 64 (more
than enough to drive the 20 thus there are some
extra words. SUPPRESSOR MUTATIONS (counteract
another mutation or suppress the mutation) 1st
demonstration of triplets. T4 rII system
proflavin mutants (delete or insert 1
base) Example of Insertion ATCTTA ATCGTTA
to give ATCGTTAExample of Deletion ATCTTA
AT_TTA to give ATTTA
Treat rIIx mutants with proflavin again, plate on
E.coli K a very few plaques appeared to be wt
revertants these behaved as wt rII phage
however, when additioal genetic crosses were done
with the T4 system, it was shown that a 2nd
mutation had been created that suppressed the
first one. Suggested that the reading frame was
shifted by an insertion (rIIx) and then shifted
back by a deletion (or vice versa?)
rII mutant x cannot plate on K
NOT like WT!!
The proflavin induced mutants were in fact
FRAMESHIFT MUTATIONS
5
Frameshifts shift the reading frame by
insertion/deletion of a single base. ATA ATC TTT
TTA insertion of a G after C now gives ATA ATC
GTT TTT A and ORF (open reading frame) is
shifted Another example THE FAT CAT ATE THE BIG
RAT delete the C in CAT THE FAT ATA TET HEB IGR
AT Non-sense downstream of the deletion In
the rII system of suppressors, here is what
happened WT CAU CAU CAU CAU CAU Insert an A
in 2nd codon CAU ACA UCA UCA UCA U
Shift in the reading frame nonsense mRNA
results and defective protein Suppressor of above
possible with a deletion mutation CAU ACA UCU
CAU CAU Restores the reading frame but still
not quite right CAU CAU CAU CAU CAU WT sequence
compared in green, 2 amino acids changed
6
FROM SUPPRESSOR MUTATIONS.If the original FCO is
defined as plus and the suppressor as minus
it was shown that a could suppress a but
could never suppress another . BUT three
pluses always gave back WT as did three
minuses! THE GENETIC CODE APPEARS TO BE A TRIPLET
OF 3 NUCLEOTIDES! PROOF of a triplet code from
the sequence level, 3 deletions in a single gene
will restore the reading frame (as will 3 single
base insertions) CAU CAU CAU CAU CAU CAU CAU
delete orange bases gives CAU ACA UAU CAU CAU
CAU WT MUT MUT WT WT
WT The findings were confirmed at the amino acid
sequence level. Also the code was shown to be
degenerate. Most amino acids have more than one
defined codon (triplet). Thus, some are
specified by gt1 codon.
7

• Cracking the genetic code
• Used polynucleotide phosphorylase to synthesize
  mRNA (NOT DNA dependent)Simple ss RNA synthesis
  driven by base proportions.
• EX add only UTP poly U.
• Nirenberg expts. poly U programs synthesis of a
  polyptn (phe)mix different ratios of
  XTPChemically analyze amino acid content
  (ratios) in the ptn product based on expected
  frequencies
• Could deduce specific sequence related to the
  triplet code.

8
How do triplets attract a specific aa? (3 BP
bonding pretty weak). tRNA adaptor molecules
recognize the triplet and bring aa to the mRNA
for TLN. The t in tRNA is transfer. rThe
tRNAs are encoded by genome. Amino acids are
illiterate and require the tRNA to mediate
insertion.
A general model tRNA 3 end left
Features -3D shape flat cloverleaf-anticodon
loop region -each tRNA unique folded structure
reqqd to recognize tRNA synthetase tRNA syn
joins tRNA (as amino acylated form) to specific
amino acid).
Note of Modified bases methy G, dimethylG,
methylI, pseudoU
9
Complete code worked out with mini-mRNA (3
residues). Too small to drive tln but could bind
aminoacylated tRNAs ( fine tuning possible)
Some key findings codons exceeds aa (64 vs.
20) Some aa have as many as 6 codons (ser),
others only 1 thus there are 6 ser tRNAs Some
tRNAs can bring in a specific aa using DIFFERENT
codonsdue to Wobble Wobble the 3rd base is
the wobble base (5) in the anticodon. There is
some slop in rules of C here. Stop Codons
specify termination. Have names like amber
(UAG), opal (UGA) and ochre. Insertion of a
stop a nonsense mutation (never bind to
aminoacylated tRNA.
10
Protein Synthesis
11

• The overall chemical reaction
• AA binding to a specific tRNA AA1 plus tRNA1
  ATP aa-tRNA1 AMP Ppithis charges the
  tRNA also note each aa has its own tRNA and
  synthetase
• Energy of charged tRNA drives peptide bond
  formation by linking one residue to another at
  ribosome.
• Requires mRNA, tRNA, rRNA, ribosomes, ptn
  cofactors/subunits, misc. enzyme (synthetases and
  others) salts, ions, aa pools, etc.

Synthetase1
Peptidyl transferaseat ribosome
aa1
-tRNA1
aa2
aa1
-tRNA2
-tRNA2
- aa2
tRNA1
Continue to add aa
Ribosomes 2 subunits in bacteria, 50s/30s (
70s assembly) (euk 40s/60s 80s)
12
Peptide syn. On the ribosome Overview. -mRNA
binds 30s, charged tRNAs at P and A site. P site
site of growing chain A site, entry of new
charged tRNA-aa residue Chain elongated by
transfer of nascent chain onto charged, incoming
tRNA-aa Deacetylate tRNA exits, leaves A site
vacant for next tRNA-aa

• Read over
• Process of
• Initiation
• Elongation
• Termination
• movies on web, Fig. 10-39

13

• Key points to review and know
• Initiation
• Init. Factors
• Init. tRNA (tRNAfMET). Codon AUG, GUG or UUG
  init. Codons.
• First step mRNA binds 30s sub (stimulated by
  IF3) ribosomes free until engaged in TLN
• 1. How to pick out correct AUG or GUG??
  Ribosome- binding mediated by S-D sequence.
  Real AUGs have the 16s S-D site located upstream
  that binds to 3 end of ribosome (30s).
• Second step IF2/GTP/fMet-tRNA interaction at P
  site
• Third GTP energy drives the assy of complete
  70s ribosom.

14
NEXT Elongation. Factors EF-Tu, EF-T and EF-G
mediate the processEF-Tu entry EF-Ts,
release etc. GTP hydrolysis provides the
energy. FINALLY Termination and release stop
codons (UAG) recognized by the release
factors CONCEPT of nonsense suppressors .
Recall nonsense mutation inserts a stop. Many
nonsense suppressors mutations in tRNA
anticodons may allow recognition of some stop
codons and insertions of an aa residue, thereby
allows continuation of chain. Does this mess up
all stops in the cell? Generally no since many
normal stops contain several (or at least 2) stop
codons in a row. This coupled with fact that
non-sense suppressors are probably inefficient,
allows for normal tln to occur
15
(No Transcript)
16

• I. Post-Tln processing of proteins
• -some ptns secreted (a leader sequence called a
  signal sequence might be added at N-terminus.
  hydrophobic 5-25 aa long SEE FIG 10-41
• -some are phosphorylated, acetylated, methylated,
  etc.
• PTN Splicing intervening ptn sequences (IVPS)
  removed or spliced out like RNA autocataltyic.
  Note that all IVPS contain endonuclease activity
• II. Universality of the code Appears common to
  all organisms with minor differences in
  mitochondria (see text)
• Also TLN seems extremely well conserved.
• In vitro Tln can be programmed by foreign
  mRNA.
• In vivo Tln (microinject mRNA into cytoplasm)
  can as well.