Berg J.M., Tymoczko J.L., Stryer L. Biochemistry
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Protein Factors Play Key Roles in Protein Synthesis
Protein synthesis takes place in three phases: initiation, elongation, and termination. In prokaryotes, mRNA,
formylmethionyl-tRNA
f
(the special initiator tRNA that recognizes AUG), and a 30S ribosomal subunit come together
with the assistance of initiation factors to form a 30S initiation complex. A 50S ribosomal subunit then joins this
complex to form a 70S initiation complex, in which fMet-tRNA
f
occupies the P site of the ribosome.
Elongation factor Tu delivers the appropriate aminoacyl-tRNA to the ribosome A (aminoacyl) site as an EF-Tu ·
aminoacyl-tRNA · GTP ternary complex. EF-Tu serves both to protect the aminoacyl-tRNA from premature cleavage
and to increase the fidelity of protein synthesis by ensuring that the correct codon-anticodon pairing has taken place
before hydrolyzing GTP and releasing aminoacyl-tRNA into the A site. Elongation factor G uses the free energy of GTP
hydrolysis to drive translocation. Protein synthesis is terminated by release factors, which recognize the termination
codons UAA, UGA, and UAG and cause the hydrolysis of the ester bond between the polypeptide and tRNA.
Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in
Translation Initiation
The basic plan of protein synthesis in eukaryotes is similar to that of prokaryotes, but there are some significant
differences between them. Eukaryotic ribosomes (80S) consist of a 40S small subunit and a 60S large subunit. The
initiating amino acid is again methionine, but it is not formylated. The initiation of protein synthesis is more complex in
eukaryotes than in prokaryotes. The AUG closest to the 5
end of mRNA is nearly always the start site. The 40S
ribosome finds this site by binding to the 5
cap and then scanning the RNA until AUG is reached. The regulation of
translation in eukaryotes provides a means for regulating gene expression. Many antibiotics act by blocking prokaryotic
gene expression.
Key Terms
translation
ribosome
transfer RNA (tRNA)
codon
anticodon
aminoacyl-tRNA synthetase
50S subunit
30S subunit
polysome
Shine-Dalgarno sequence
peptidyl transferase center
wobble hypothesis
initiation factor
elongation factor
elongation factor Tu (EF-Tu)
elongation factor Ts (EF-Ts)
elongation factor G (EF-G)
molecular mimicry
release factor
III. Synthesizing the Molecules of Life 29. Protein Synthesis
Problems
1.
Synthetase mechanism. The formation of isoleucyl-tRNA proceeds through the reversible formation of an enzyme-
bound Ile-AMP intermediate. Predict whether
32
P-labeled ATP is formed from
32
PP
i
when each of the following sets
of components is incubated with the specific activating enzyme:
(a) ATP and
32
PP
i
(b) tRNA, ATP, and
32
PP
i
(c) Isoleucine, ATP, and
32
PP
i
See answer
2.
Light and heavy ribosomes. Ribosomes were isolated from bacteria grown in a "heavy" medium (
13
C and
15
N) and
from bacteria grown in a "light" medium (
12
C and
14
N). These 60S ribosomes were added to an in vitro system
actively engaged in protein synthesis. An aliquot removed several hours later was analyzed by density-gradient
centrifugation. How many bands of 70S ribosomes would you expect to see in the density gradient?
See answer
3.
The price of protein synthesis. What is the smallest number of molecules of ATP and GTP consumed in the synthesis
of a 200-residue protein, starting from amino acids? Assume that the hydrolysis of PP
i
is equivalent to the hydrolysis
of ATP for this calculation.
See answer
4.
Contrasting modes of elongation. The two basic mechanisms for the elongation of biomolecules are represented in
the adjoining illustration. In type 1, the activating group (X) is released from the growing chain. In type 2, the
activating group is released from the incoming unit as it is added to the growing chain. Indicate whether each of the
following biosyntheses is by means of a type 1 or a type 2 mechanism:
(a) Glycogen synthesis
(b) Fatty acid synthesis
(c) C
5
C
10
C
15
in cholesterol synthesis
(d) DNA synthesis
(e) RNA synthesis
(f) Protein synthesis
See answer
5.
Suppressing frameshifts. The insertion of a base in a coding sequence leads to a shift in the reading frame, which in
most cases produces a nonfunctional protein. Propose a mutation in a tRNA that might suppress frameshifting.
See answer
6.
Tagging a ribosomal site. Design an affinity-labeling reagent for one of the tRNA binding sites in E. coli ribosomes.
See answer
7.
Viral mutation. An mRNA transcript of a T7 phage gene contains the base sequence
Predict the effect of a mutation that changes the G marked by an arrow to A.
See answer
8.
Two synthetic modes. Compare and contrast protein synthesis by ribosomes with protein synthesis by the solid-phase
method (see Section 4.4).
See answer
9.
Enhancing fidelity. Compare the accuracy of (a) DNA replication, (b) RNA synthesis, and (c) protein synthesis.
Which mechanisms are used to ensure the fidelity of each of these processes?
See answer
10.
Triggered GTP hydrolysis. Ribosomes markedly accelerate the hydrolysis of GTP bound to the complex of EF-Tu
and aminoacyl-tRNA. What is the biological significance of this enhancement of GTPase activity by ribosomes?
See answer
11.
Blocking translation. Devise an experimental strategy for switching off the expression of a specific mRNA without
changing the gene encoding the protein or the gene's control elements.
See answer
12.
Directional problem. Suppose that you have a protein synthesis system that is actively synthesizing a protein
designated A. Furthermore, you know that protein A has four trypsinsensitive sites, equally spaced in the protein,
that, on digestion with trypsin, yield the peptides A
1
, A
2
, A
3
, A
4
, and A
5
. Peptide A
1
is the amino-terminal peptide,
and A
5
is the carboxyl peptide. Finally, you know that your system requires 4 minutes to synthesize a complete
protein A. At t = 0, you add all 20 amino acids, each carrying a
14
C label.
(a) At t = 1 minute, you isolate intact protein A from the system, cleave it with trypsin, and isolate the five peptides.
Which peptide is most heavily labeled?
(b) At t = 3 minutes, what will be the order of labeling of peptides from greatest to least?
(c) What does this experiment tell you about the direction of protein synthesis?
See answer
13.
Translator. Aminoacyl-tRNA synthetases are the only component of gene expression that decodes the genetic code.
Explain.
See answer
14.
A timing device. EF-Tu, a member of the G-protein family, plays a crucial role in the elongation process of
translation. Suppose that a slowly hydrolyzable analog of GTP were added to an elongating system. What would be
the effect on rate of protein synthesis?
See answer
Mechanism Problems
15.
Molecular attack. What is the nucleophile in the reaction catalyzed by peptidyl transferase? Write out a plausible
mechanism for this reaction.
See answer
16.
Evolutionary amino acid choice. Ornithine is structurally similar to lysine except ornithine's side chain is one
methylene group shorter than that of lysine. Attempts to chemically synthesize and isolate ornithinyl-tRNA proved
unsuccessful. Propose a mechanistic explanation. (Hint: Six-membered rings are more stable than seven-membered
rings).
See answer
Chapter Integration Problems
17.
Déjà vu. Which protein in G-protein cascades plays a role similar to that of elongation factor Ts?
See answer
18.
Family resemblance. Eukaryotic elongation factor 2 is inhibited by ADP ribosylation catalyzed by diphtheria toxin.
What other G proteins are sensitive to this mode of inhibition?
See answer
Data Interpretation Problem
19.
Helicase helper. The initiation factor eIF4A displays ATPdependent RNA helicase activity. Another initiation
factor, eIF4H, has been proposed to assist the action of eIF4A. Graph A shows some of the experimental results
from an assay that can measure the activity of eIF4A helicase in the presence of eIF4H.
(a) What are the effects on eIF4A helicase activity in the presence of eIF4H?
(b) Why did measuring the helicase activity of eIF4H alone serve as an important control?
(c) The initial rate of helicase activity of 0.2 µM of eIF4A was then measured with varying amounts of eIF4H
(graph B). What ratio of eIF4H to eIF4A yielded optimal activity?
(d) Next, the effect of RNA-RNA helix stability on the initial rate of unwinding in the presence and absence of
eIF4H was tested (graph C). How does the effect of eIF4H vary with helix stability?
(e) How might eIF4H affect the helicase activity of eIF4A?
[Data after N. J. Richter, G. W. Rodgers, Jr., J. O. Hensold, and W. C. Merrick, 1999. Further biochemical and
kinetic characterization of human eukaryotic initiation factor 4H. J. Biol. Chem. 274:35415
35424.]
See answer
Media Problem
20.
Same difference? Thr-tRNA synthetase (class II) contains an editing site that recognizes and hydrolyzes
misacylated SertRNA
Thr
. This proofreading ability is needed because serine can fit into and make almost as
many favorable interactions with the aminoacylation site as threonine, allowing Thr-tRNA synthetase to mistakenly
couple serine to threonyl-tRNA. Which aminacyl-tRNA synthetase is most likely to have a comparable need to
proofread and edit tRNAs misacylated with valine? Given the class of aminoacyl-tRNA synthetases to which this
enzyme belongs, how similar do you expect its editing site to be to that of Thr-tRNA synthetase?
III. Synthesizing the Molecules of Life 29. Protein Synthesis
Selected Readings
Where to start
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P. Schimmel and L. Ribas de Pouplana. 2000. Footprints of aminoacyl- tRNA synthetases are everywhere Trends
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Transfer RNA
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Ribosomes and ribosomal RNAs
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N. Ban, P. Nissen, J. Hansen, P.B. Moore, and T.A. Steitz. 2000. The complete atomic structure of the large ribosomal
subunit at 2.4 Å resolution Science 289: 905-920. (PubMed)
A.P. Carter, W.M. Clemons, D.E. Brodersen, R.J. Morgan-Warren, B.T. Wimberly, and V. Ramakrishnan. 2000.
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Initiation factors
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J.H. Lee, S.K. Choi, A. Roll-Mecak, S.K. Burley, and T.E. Dever. 1999. Universal conservation in translation initiation
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(PubMed)
Elongation factors
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Termination
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III. Synthesizing the Molecules of Life
30. The Integration of Metabolism
We have been examining the biochemistry of metabolism one pathway at a time, but in living systems many pathways
are operating simultaneously. Each pathway must be able to sense the status of the others to function optimally to meet
the needs of an organism. How is the intricate network of reactions in metabolism coordinated? This chapter presents
some of the principles underlying the integration of metabolism in mammals. We begin with a recapitulation of the
strategy of metabolism and of recurring motifs in its regulation. We then turn to the interplay of different pathways in
regard to the flow of molecules at three key crossroads: glucose 6-phosphate, pyruvate, and acetyl CoA. We consider the
differences in the metabolic patterns of the brain, muscle, adipose tissue, kidney, and liver. Finally, we examine how the
interplay between these tissues is altered in a variety of metabolic perturbations. These discussions will illustrate how
biochemical knowledge illuminates the functioning of the organism.
III. Synthesizing the Molecules of Life 30. The Integration of Metabolism