Protein Synthesis

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Lecture 15

• Protein Synthesis

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Lecture outline

• Roles of RNA and the genetic code
• Aminoacylation of tRNAs
• Translation
• initiation _at_
• elongation_at_
• termination_at_

fidelity _at_ rate
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The roles of RNA in protein synthesis
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The genetic code triplet and redundant
How was the code cracked?
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The genetic code is (almost) universal
Exceptions to the genetic code.
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tRNAs
Link codons to amino acids. 30 - 100 tRNA genes
per cell. 73 - 93 nt. long. Many post-Tx
modifications polycistronic primary transcript
(RNaseP, RNaseD). many unusual bases (5 -
15) Strong secondary structure (50
double-stranded).
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tRNA PROCESSING
Removal of 5 leader and 3 trailer order not
absolute CCA may be encoded (prok.) or added
post-transcriptionally (euk.) Acceptor stem
sometimes edited Some tRNAs have introns in the
anticodon loop Many nucleotide modifications
editing intron
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Modified bases in tRNA
Alberts et al. Molecular Biology of the cell
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tRNA structure
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Linking tRNA to amino acid...
is done by aminoacyl tRNA synthetases
(AARS) 20 amino acids 30 - 100 different
tRNAs 64 codons (3 stops) 20 aminoacyl tRNA
synthetases Each AARS is specific for an amino
acid. Can bind multiple tRNAs. Each tRNA is
recognized by a specific AARS.
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(No Transcript)
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The aminoacyl-tRNA synthetase reactions
!!!!
Each tRNA molecule is recognized by a specific
aminoacyl-tRNA synthetase
!!!!
!!!! proofreading
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Hydrolytic Editing
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Translation is a two-step decoding process
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Nonstandard base pairing between codons and
anticodons -- the wobble hypothesis
First two bases of a codon pair in the standard
way.
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The nonstandard, wobble base pairs
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Ribosomes
The enzyme of protein synthesis. 70 RNA (3
different RNAs). 50 - 80 different proteins. 2.7
x 106 daltons 200 angstroms. The most abundant
RNA/protein complex in cells. Must accommodate
mRNAs, tRNAs and the growing peptide chain. The
rRNAs are catalytically active. 3 - 5 AAs per
second (small proteins in lt1 minute).
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Ribosome structure in prokaryotes eukaryotes
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Viva la difference!
In prokaryotes, transcription and translation are
coupled. In eukaryotes, RNA is processed
(capped, spliced, polyadenylated) and transported
from the nucleus before it is translated.
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The start codon is recognized by
methionyl-tRNAiMet
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Translation of mRNA
Alberts et al. Molecular Biology of the Cell
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Translation initiation - where to start?
Prokaryotes the Shine-Dalgarno sequence AAGGAGG
consensus 5 - 13 nt. from start codon (AUG, GUG,
UUG) Internal initiations common. Eukaryotes
the scanning mechanism 5 of mRNA is recognized
(the m7G cap) First in-context AUG is
used. Context GCCACCaugG Internal initiations
are very controversial.
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The bacterial initiation complex
mRNA Small ribosomal subunit Initiation factor 2
(IF-2) and GTP tRNAiMet IF-1 and IF-3 are also
required. Then IF-3 is released and the large
ribosomal subunit is recruited.
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Initiation of bacterial protein synthesis
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Initiation of eukaryotic protein synthesis
Internal initiations are controversial
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Initiation of translation in eukaryotes
Lodish, Molecular Cell Biology
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Intiation of translation in eukaryotes -2
Lodish, Molecular Cell Biology
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eIF-2 is a translation control point in eukaryotes
Phosphorylated eIF-2 cannot release GDP, stopping
translation. eIF-2 is phosphorylated during
stationary phase, starvation etc. Various viruses
interfere with this regulation Herpes (HSV) -
eIF-2 - phosphatase Adenovirus (Ad) makes a
decoy competitor RNA Hepatitis C blocks
activation of the eIF-2 kinase.
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Elongation
Aminoacylated tRNAs are brought to the ribosome
by elongation factor Tu (EF-Tu) EF-Tu is very
abundant (5 of total protein). protects
aa-tRNAs from spontaneous hydrolysis. GTP-binding
protein GTP hydrolysis, EF-Ts control EF-Tu
function.
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The EF-Tu reaction cycle
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Translation elongation
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Accuracy of protein synthesis
The real rate is 1 x 10-4
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Termination of translation in eukaryotes
Lodish et al. Molecular Cell Biology
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Release factors recognize stop codons
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Speeding up translation
Elongation proceeds at 3- 5 AAs per
second. Protein synthesis is enhanced by One
mRNA, many ribosomes (polysomes). Ribosome
recycling
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Ribosome recycling
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Many antibiotics inhibit translation