Biology 212 General Genetics
Lecture 19: Gene Expression II (Protein Synthesis)
Fall 2006
Reading: Chap. 8 pp. 293-309
Lecture Outline:
1. Key components in protein synthesis
2. Process of protein synthesis
3. Genetic evidence for the triplet code
4. Role of tRNA
Lecture:
1. Key components in protein synthesis
A. mRNA=messenger RNA; carries the code that determines the amino acid sequence
for a protein
B. ribosomes=particles composed of rRNAs and proteins that are the sites of protein
synthesis
 Eukaryotic ribosome: 40S + 60S  80S ribosome
C. tRNA=transfer RNA; carries amino acid to ribosome, contains triplet anticodon that
pairs with codon in mRNA
D. aminoacyl-tRNA synthetases="charging enzymes" that add a specific amino acid to
its cognate tRNA
E. initiation factors, elongation factors and termination factors: proteins that assist
with stages of protein synthesis
Some differences prokaryotes vs. eukaryotes
Prokaryotes
Transcription and translation both occur in
cytoplasm; said to be "coupled"
Eukaryotes
Transcription and RNA processing occur in
nucleus, mRNA exported to cytoplasm
Translation occurs in cytoplasm
mRNAs generally encode for only one
protein
Many mRNAs are polycistronic=encode
for more than one protein
2. Process of protein synthesis (eukaryotes)
Three major phases: initiation, elongation, and termination
Fig. 8-15 (initiation)
i) initiation: mRNA binds ribosome
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

initiation factors (eIFs = eukaryotic initiation factors) bind to 5' cap on mRNA
initiator tRNA (charged met tRNA), additional initiation factors and the small
(40S subunit) then bind to form the initiation complex
ii) elongation: extend polypeptide chain


elongation factors and 60S ribosomal subunit are recruited to the ribosome
initiation factors are released
3 main steps are repeated:
(1) each new aminoacylated tRNA is brought to the ribosome
(2) the new peptide bond is formed: catalysis by the RNA component of ribosome
(3) the ribosome moves to the next codon along the mRNA (5'  3') = translocation
Fig. 8.16



multiple repeated cycles of elongation until a termination codon is reached
energy to drive protein synthesis comes from hydrolysis of GTPGDP
protein chain grows from the amino terminus  carboxy terminus
iii) termination: protein synthesis is halted


when a stop codon (UAA, UAG, UGA) is reached, a release factor binds the
ribosome
GTP hydrolysis provides energy to cleave the polypeptide from the tRNA, to
eject the release factor, and to dissociate the 40S and 60S subunits of the
ribosome
Summary of information transfer:
5'
3'
transcription
3'
5'
5'
translation
3' mRNA
H2N
DNA (template strand)
COOH
3. Genetic evidence for the triplet code
Experiments of Francis Crick and Sidney Brenner
1961
T4: virus of E. coli
r: rapid lysis mutant; forms a different appearing plaque from wild type
rII: mutations in the rapid lysis gene II locus
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i) induce mutations with proflavin
rII+  rII


"intercalating agent"= sits between stacked base pairs on DNA
when DNA is replicated in the presence of proflavin, tend to get insertions or
deletions of one to a few nucleotides
insertions or deletions of one or two nucleotides typically cause "frameshift"
mutations that shift the reading frame, often a stop codon is encountered in the
new reading frame
Analysis of frameshift mutations: Fig 8.25
+/- 1 nucleotide:
+/- 2 nucleotides:
+/- 3 nucleotides:
get frameshifts
get frameshifts
often not harmful; reading frame restored
ii) back mutation (reversion)
rII-  rII+
these often result from a mutation that restores the reading frame
4. Role of tRNA and codon redundancy


the genetic code has evolved to minimize the effects of mutation
tRNAs play several key roles in determining the accuracy of protein synthesis
Role of tRNA:
 High specificity of activating enzyme for its cognate tRNA
 Activating enzyme binds anticodon and 3' terminus of tRNA (-CCA) sequence
 If added amino acid is not consistent with the anticodon, the enzyme reverses the
reaction and the amino acid is released
Wobble pairing and codon degeneracy:
Example: codon for his
Codon
5' CAC 3'
5' CAU 3'
Anticodon
3' GUG 5'
3'GUG 5'
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


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Third position of the codon is not bound as tightly by the tRNA as the first and
second positions.
Same tRNA can often read 2 codons. The G in the anticodon can pair with C or
U in the third position of the codon.
Wobble hypothesis: Developed by Francis Crick in 1966. Concept that the first 2
bp of codon-anticodon pair determines specificity for amino acid
Explains why 61 codons for amino acids can be translated with only 38 different
tRNAs
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