Translation
mRNA triplet code read by read by ribosome and aminoacyl tRNAs and translated into peptide
tRNA
each amino acid covalently linked to at least one cognate tRNA
Getting right amino acid on tRNA with right anticodon
aminoacyl tRNA synthetases (RS)
specific synthetase for each amino acid
attaches proper amino acid residue to 3 end of tRNA (sometimes to adjacent 2-OH)
energetically unfavorable-uses ATP and aminoacyl adenylate intermediate to drive reaction
aa + ATP --> aminoacyl AMP + PPi --> aminoacyl AMP + 2Pi
aminoacyl AMP + tRNA --> aminoacyl-tRNA + AMP
2 coupled reaction catalyzed by the same enzyme
aminoacyl AMP intermediate never exposed to solvent
e. g. tryosyl tRNA syn. (TyrRS)
binds substrates through 12 H-bonds
catalysis accelerated by stabilization of transition state:
2 H-bonds to gamma PO4 of ATP favor trigonal bypryamid transition state at alpha PO4
Mutate -->10e6-fold less efficient
Recognition of correct amino acid
most amino acids very different, but some vary from each other in subtle ways
e.g. valine, isoleucine and threonine
Val and Ile binding to IleRS:
extra CH2 group of Ile ~-3kcal/mol binding energy
200-fold more likely to bind Ile
but intracellular concentration of Val 5-fold higher
only 40-fold theoretical preference for Ile
but actual is 3000-fold. How???
synthetases have proofreading function:
hydrolytic site to hydrolyze wrong acyl-AMP before conjugation to tRNA
Ile fits best into acylation site, Val fits best in hydrolytic site
IleRS will make Val-AMP, but upon tRNA binding, Val-AMP is specifically hydrolyzed to Val + AMP
Thr and Val same size, but thr is polar. ValRS has polar hydrolytic site
Recognition of tRNA by synthetases
2 classes of synthetases recognize different features of tRNA substrate
have structurally different active sites
Class I |
Class II |
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Gln (I) |
Asp(II) |
generally RS for larger amino acids |
more ancient origin |
Many of both classes of synthetases recognize bases of the anticodon, but some do not.
Instead they recognize the variable loop or specific base-pairs of the acceptor stem
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II |
II |
I |
II |
Exceptional cases:
Some organisms do not have all 20 RS:
lack
of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles.
tRNAGln is misacylated with Glu, then conjugated Glu converted to Gln by transamidation reaction. Only amine transferred, not exchange of entire amino acid. In archaea, similar system for Asp and Asn
mRNA translated at the ribosome
each codon of mRNA recognized by base-pairing with anticodon of appropriate aminoacyl tRNA --> THE GENETIC CODE
3 residues/codon
based on genetic studies of insertion and deletion mutants
Genetic code elucidated in the 1950s and 60s when you couldnt synthesize long unique sequence RNAs.
ribosome binds appropriate aminoacyl tRNA in the presence of trinucleotide "mRNA"
50 of 64 codons determined this way
rest were determined by synthesizing homopolymers or RNAs consisting of di- tri- or tetranucleotide repeats.
these could be used as translation templates by ribosomes under non-physiological conditions even though the RNAs did not have the proper initiation signals:
RNA products
polyU poly(Phe)
polyA poly(Lys)
polyC poly(Pro)
poly(AC) poly(ThrHis)
e.g.:
ACA|CAC|ACA|CAC|ACA|CAC|ACA|CAC|
Thr-His-Thr-His-Thr-His-Thr-His-
poly(AG) -->
poly(ArgGlu)
poly(UC) -->
poly(SerLeu)
poly(UUC) -->
poly(Phe) + poly(Ser) + poly(Leu)
e.g.:
UUC|UUC|UUC|UUC|UUC|UUC|UUC|UUC|
Phe-Phe-Phe-Phe-Phe-Phe-Phe- or
U|UCU|UCU|UCU|UCU|UCU|UCU|UCU|UCU|
Ser-Ser-Ser-Ser-Ser-Ser-Ser-Ser- or
UU|CUU|CUU|CUU|CUU|CUU|CUU|CUU|CUU
Leu-Leu-Leu-Leu-Leu-Leu-Leu-Leu-
poly(AAG) -->
poly(Lys) + poly(Arg) + poly(Glu)
poly(UUG) -->
poly(Leu) + poly(Cys) + poly(Val)
poly(CCA) -->
poly(His) + poly(Pro) + poly(Thr)
poly(GUA) -->
poly(Val) + poly(Ser)
poly(UAC) -->
poly(Tyr) + poly(Thr) + poly(Leu)
poly(AUC) -->
poly(Ile) + poly(Ser) + poly(His)
poly(GAU) -->
poly(Asp) + poly(Met)
poly(UAUC) --> TyrLeuSerIleTyrLeuSerIle
UAU|CUA|UCU|AUC|UAU|CUA|UCU|AUC
Tyr-Leu-Ser-Ile-Tyr-Leu-Ser-Ile
but poly(GUAA) --> only di- and tripeptides
GUA|AGU|AAG|UAA|GUA|AGU|AAG|UAA|GUA|A
Val-Ser-Lys-STOP
Together these results were used to piece together the code:
First |
Second U |
C |
A |
G |
Third |
U
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UUU Phe |
UCU Ser |
UAU Tyr |
UGU Cys |
U |
C |
CUU Leu |
CCU Pro |
CAU His |
CGU Arg |
U |
A |
AUU Ile |
ACU Thr |
AAU Asn |
AGU Ser |
U |
G |
GUU Val |
GCU Ala |
GAU Asp |
GGU Gly |
U |
doesnt require 64 different tRNAs due to ability of anticodon to pair with more than one codon by Wobble pairing:
e.g. G can pair with C or U
anticodon | anticodon | |
3 AAG 5 | or | 3 AAG 5 |
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5 UUU 3 | 5 UUC 3 | |
codon | codon |
alanine tRNA CGI anticodon:
anticodon | anticodon | anticodon | ||
3 CGI 5 | or | 3 CGI 5 | or | 3 CGI 5 |
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5 GCU 3 | 5 GCC 3 | 5 GCA 3 | ||
codon | codon | codon |
Bacterial ribosome
2700 kdaltons 200Å in diameter
sediments at 70S
can be dissociated into large and small subunits
A. Large 50S:
contains 23S and 5S RNAs
plus 34 proteins
B. Small 30S:
contains 16S RNA 21 proteins
Cate et al (1999) Science 285, 2095-2104
Translation initiation:
fMet-tRNA: special prokaryotic initiator tRNA acylated by normal MetRS
internal methionines charged onto different tRNA using same RS
The 30S subunit binds initiation factor IF3 and mRNA
IF3 prevents premature association of 50S subunit
mRNA is bound by base-pairing with 3 end of 16S rRNA
the mRNA sequence which binds is called the Shine-Delgarno sequence
found within 10 nts of the initiation codon (usually AUG, sometimes GUG)
Initiation codons are recognized by their proximity to the Shine-Delgarno sequence in prokaryotes. This defines the reading for translation
IF3-mRNA-30S complex binds to complex of IF2-GTP-tRNAfMet
--> 30S initiation complex
IF1 stimulates IF2 and IF3
The 50S subunit binds, causing IF2 to hydrolyze GTP, releasing IF3 and IF2 from the complex -->
70S initiation complex
3 sites for tRNA binding on ribosome:
P peptidyl
A amino
E exit
fMet-tRNA binds to P site
Elongation
aminoacyl tRNA for next codon delivered from RS to ribosome by
EF-Tu elongation factor
EF-TuGTP complex binds aa-tRNA and delivers it to A site of ribosome
GTP is hydrolyzed and EF-Tu dissociates from ribosome
EF-Tu in turn is bound by another factor, EF-Ts allowing release of GDP
Binding of GTP to Tu-TS complex causes release of EF-Ts and regeneration of EF-TuGTP
EF-Tu protects aa-tRNA from hydrolysis
Also acts as timer to allow for checking of codon-anticodon pairing
GTP hydrolysis -->
conformational change
codon-anticodon pairing must be strong in both contexts to remain bound to ribosome
After EF-TUGDP is released from ribosome, peptide bond is formed by peptidyl transferase activity of ribosome, which resides in 23S rRNA
Reaction mechanism of protein elongation
after peptide bond is formed, the interaction of tRNAs with 50S subunit changes:
the deacylated tRNA moves to the E site of 50S but remains in P site of 30S
the dipeptidyl tRNA moves to the P site of 50S but remains in A site of 30S
elongation factor EF-G is a translocase that acts as an engine to move the tRNA-mRNA complex with respect to the 30S subunit.
EF-GGTP binds to 50S subunit, hydrolyzes GTP to undergo conformational change which drives tRNA translocation
Once tRNAs are moved, A site is empty and ready for next aa-tRNA, P site contains growing peptidyl-tRNA ready for further elongation, the deacylated tRNA leaves the E site during translocation.
As a result, the tRNAs never lose contact with at least one ribosomal subunit, preventing the peptidyl-tRNA from diffusing away from ribosome
Peptide is synthesized in N -->
C direction, reading mRNA in 5´ -->
3´ direction
In vitro translation system:
Add 3H-leucine, incubate for times shown
Digest product with protease to map location in product
3H-leucine added to C-term of peptides already being synthesized
at early times
At later times see incorporation at N-term when later rounds of initiation occur
Termination
When a stop codon is encountered, no tRNA can bind to this codon, leaving A site empty, this allows release factors RF1 (recognizes UAA or UAG) or RF2 (UAA or UGA) to bind in the A site
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RF binding allows peptidyl transferase to hydrolyze bond between peptide and tRNA Peptide, tRNA, mRNA and RF are released and ribosome dissociates Exception: Nonsense suppression: mutant aa-tRNA with anticodon complementary to termination codon binds to A site, allowing incorporation to continue. Inhibitors for translation: Many resemble ribosome substrates, binding causes premature termination of protein synthesis
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Comparison of translation factors
in pro- vs. eukaryotes
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Prok |
Euk |
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IF2-fMet-tRNA |
eIF2-cap |
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EF-Tu+Ef-Ts |
eEF-Tu |
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EF-G |
eEF-2 |
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RF1,2,3 |
eRF |
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