LECT 20: PROTEIN SYNTHESIS AND TRANSLATIONAL CONTROL

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LECT 20 PROTEIN SYNTHESIS AND TRANSLATIONAL
CONTROL
High fidelity of protein synthesis from mRNA is
essential. Mechanisms controling translation
accuracy include very high fidelity of (1)
tRNA charging with correct amino acid (2)
codon-directed delivery of correct charged tRNA
to ribosome Mechanisms for translation
elongation and termination are very similar in
prokaryotes and eukaryotes, but mechanisms of
initiation are fundamentally
different Translation of eukaryotic mRNAs can be
regulated at the step of initiation by multiple
mechanisms Secreted and membrane-spanning
proteins are co-translationally delivered
across membranes by translocons guided by
signal and anchor sequences in the nascent
polypeptide chain
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Transfer RNA Structure and Functional Domains
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Chemistry of tRNA Charging with Amino Acid
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tRNA Synthetase Achieves Fidelity Through
Substrate Specificity and Editing
A tRNA synthetase recognizes a specific tRNA by
interaction with unique structural features of
that tRNA, including the anticodon loop
sequence. The tRNA synthetases active site
also binds correct amino acid, but sometimes
also a related amino acid. Charging with the
wrong related amino acid is uncharged by a
separate editing domain.
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Ribosome Composition
Prokaryotes
Eukaryotes
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Translation Initiation Complex in Prokaryotes
Initiation factors deliver initiator fMet-tRNA
to mRNA initiation codon positioned at the P
site of the 30S ribosomal subunit. Initiatio
n codon is AUG preceded by Shine-Delgarno
sequence that is recognized by the 16S rRNA in
the 30S ribosomal subunit.
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Translation Initiation Complex in Eukaryotes
Initiation factors deliver 40S ribosomal subunit
and Met-tRNA to 5 methylated cap on mRNA. The
40S subunit then scans down mRNA and docks
at first AUG codon. Initiation factor
interaction with 5 cap is facilitated by prior
recruitment of initiation factor to the mRNA
via polyA binding protein (PABP) bound to the
mRNAs 3 polyA tail.
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Dormant Eukaryotic mRNAs and Their Activation
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Elongation Sequentially Adds Amino Acids to a
Peptidyl-Acyl tRNA
Peptide(n)-acyl-tRNA Aminoacyl-tRNA ---gt
tRNA(uncharged) Peptide(n 1)-acyl-tRNA
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Elongation Factors and GTP Needed for tRNA
Recruitment and Translocation
EF-Tu
GTP
GDP Pi
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Release Factors Dock Recognize Stop Codons at A
Site To Promote Termination
Upon release factor docking to stop codon, water
is used instead of amino group of charged tRNA
as nucleophile to attack acyl bond of
peptidyl-acyl-tRNA. Hydrolysis releases peptide
from tRNA and ribosome.
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Secreted Proteins Have N-Terminal Signal Sequence
Signal sequence is 10-15 residue hydrophobic
stretch near N-terminus. Signal sequence
triggers secretion mechanism, is usually cleaved
at nearby downstream small amino acid (Gly-X,
Ser-X, Ala-X)
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Nascent Signal Sequence Recruits Signal
Recognition Particle and Translocon
SRP recruitment to signal sequence arrests
translation SRP docks to andopens a translocon
on the endoplasmic reticular membrane Translation
resumes with cotranslational threading into ER
lumen Lumenal peptidase cleaves off signal
sequence Completed protein transported within
vesicles and released by exocytosis
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Transmembrane Proteins Are Cotranslationally
Inserted Into Membrane
Type I TM protein has signal sequence and
downstream hydrophobic helical transmembrane
sequence and polar stop transfer element. Type II
and III TM proteins do not have signal sequence.
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Type II and III TM Proteins Have Internal
Hydrophobic Domains That Cotranslationally
Interact with ER Translocon and Resolve In
Different Ways