Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation

Figure 29.31. Structure of a Release Factor. The structure of a eukaryotic release factor reveals a tRNA-like fold. The
acceptor-stem mimic includes the sequence Gly-Gly-Gln at its tip. This region appears to bind a water molecule,
which may be brought into the peptidyl transferase center. There it can participate in the cleavage of the peptidyltRNA ester bond, with the aid of the glutamine residue and the ribosomal catalytic apparatus.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.4. Protein Factors Play Key Roles in Protein Synthesis
Figure 29.32. Structure of Ribosome Release Factor (RRF). RRF is another protein that resembles tRNA. The α
helices of this protein mimic the tRNA structure. In contrast, in EF-G, β strands are the mimics, revealing an
independent evolutionary origin.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.5. Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis
Primarily in Translation Initiation
The basic plan of protein synthesis in eukaryotes and archaea is similar to that in bacteria. The major structural and
mechanistic themes recur in all domains of life. However, eukaryotic protein synthesis entails more protein components
than does prokaryotic protein synthesis, and some steps are more intricate. Some noteworthy similarities and differences
are as follows:
1. Ribosomes. Eukaryotic ribosomes are larger. They consist of a 60S large subunit and a 40S small subunit, which come
together to form an 80S particle having a mass of 4200 kd, compared with 2700 kd for the prokaryotic 70S ribosome.
The 40S subunit contains an 18S RNA that is homologous to the prokaryotic 16S RNA. The 60S subunit contains three
RNAs: the 5S and 28S RNAs are the counterparts of the prokaryotic 5S and 23S molecules; its 5.8S RNA is unique to
eukaryotes.
2. Initiator tRNA. In eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine. However, as in
prokaryotes, a special tRNA participates in initiation. This aminoacyl-tRNA is called Met-tRNAi or Met-tRNAf (the
subscript "i" stands for initiation, and "f" indicates that it can be formylated in vitro).
3. Initiation. The initiating codon in eukaryotes is always AUG. Eukaryotes, in contrast with prokaryotes, do not use a
specific purine-rich sequence on the 5 side to distinguish initiator AUGs from internal ones. Instead, the AUG nearest
the 5 end of mRNA is usually selected as the start site. A 40S ribosome attaches to the cap at the 5 end of eukaryotic
mRNA (Section 28.3.1) and searches for an AUG codon by moving step-by-step in the 3 direction (Figure 29.33). This
scanning process in eukaryotic protein synthesis is powered by helicases that hydrolyze ATP. Pairing of the anticodon of
Met-tRNAi with the AUG codon of mRNA signals that the target has been found. In almost all cases, eukaryotic mRNA
has only one start site and hence is the template for a single protein. In contrast, a prokaryotic mRNA can have multiple
Shine-Dalgarno sequences and, hence, start sites, and it can serve as a template for the synthesis of several proteins.
Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate. The
prefix eIF denotes a eukaryotic initiation factor. For example, eIF-4E is a protein that binds directly to the 7methylguanosine cap (Section 28.3.1), whereas eIF-4A is a helicase. The difference in initiation mechanism between
prokaryotes and eukaryotes is, in part, a consequence of the difference in RNA processing. The 5 end of mRNA is
readily available to ribosomes immediately after transcription in prokaryotes. In contrast, pre-mRNA must be processed
and transported to the cytoplasm in eukaryotes before translation is initiated. Thus, there is ample opportunity for the
formation of complex secondary structures that must be removed to expose signals in the mature mRNA. The 5 cap
provides an easily recognizable starting point. In addition, the complexity of eukaryotic translation initiation provides
another mechanism for gene expression that we shall explore further in Chapter 31.
4. Elongation and termination. Eukaryotic elongation factors EF1α and EF1β γ are the counterparts of prokaryotic EF-
Tu and EF-Ts. The GTP form of EF1α delivers aminoacyl-tRNA to the A site of the ribosome, and EF1β γ catalyzes the
exchange of GTP for bound GDP. Eukaryotic EF2 mediates GTP-driven translocation in much the same way as does
prokaryotic EF-G. Termination in eukaryotes is carried out by a single release factor, eRF1, compared with two in
prokaryotes. Finally, eIF3, like its prokaryotic counterpart IF3, prevents the reassociation of ribosomal subunits in the
absence of an initiation complex.
29.5.1. Many Antibiotics Work by Inhibiting Protein Synthesis
The differences between eukaryotic and prokaryotic ribosomes can be exploited for the development of antibiotics
(Table 29.4). For example, the antibiotic puromycin inhibits protein synthesis by causing nascent prokaryotic
polypeptide chains to be released before their synthesis is completed. Puromycin is an analog of the terminal aminoacyladenosine part of aminoacyl-tRNA (Figure 29.34).
It binds to the A site on the ribosome and inhibits the entry of aminoacyl-tRNA. Furthermore, puromycin contains an αamino group. This amino group, like the one on aminoacyl-tRNA, forms a peptide bond with the carboxyl group of the
growing peptide chain. The product, a peptide having a covalently attached puromycin residue at its carboxyl end,
dissociates from the ribosome.
Streptomycin, a highly basic trisaccharide, interferes with the binding of formylmethionyl-tRNA to ribosomes and
thereby prevents the correct initiation of protein synthesis. Other aminoglycoside antibiotics such as neomycin,
kanamycin, and gentamycin interfere with the decoding site located near nucleotide 1492 in 16S rRNA of the 30S
subunit (Section 29.3.9). Chloramphenicol acts by inhibiting peptidyl transferase activity. Erythromycin binds to the 50S
subunit and blocks translocation. Finally, cyclohexamide blocks peptidyl transferase activity in eukaryotic ribosomes,
making a useful laboratory tool for blocking protein synthesis in eukaryotic cells.
29.5.2. Diphtheria Toxin Blocks Protein Synthesis in Eukaryotes by Inhibiting
Translocation
Diphtheria was a major cause of death in childhood before the advent of effective immunization. The lethal effects
of this disease are due mainly to a protein toxin produced by Corynebacterium diphtheriae, a bacterium that grows
in the upper respiratory tract of an infected person. The gene that encodes the toxin comes from a lysogenic phage that is
harbored by some strains of C. diphtheriae. A few micrograms of diphtheria toxin is usually lethal in an unimmunized
person because it inhibits protein synthesis. The toxin is cleaved shortly after entering a target cell into a 21-kd A
fragment and a 40-kd B fragment. The A fragment of the toxin catalyzes the covalent modification of an important
component of the protein-synthesizing machinery, whereas the B fragment enables the A fragment to enter the cytosol of
its target cell.
A single A fragment of the toxin in the cytosol can kill a cell. Why is it so lethal? The target of the A fragment is EF2,
the elongation factor catalyzing translocation in eukaryotic protein synthesis. EF2 contains diphthamide, an unusual
amino acid residue of unknown function that is formed by posttranslational modification of histidine. The A fragment
catalyzes the transfer of the adenosine diphosphate ribose unit of NAD+ to a nitrogen atom of the diphthamide ring
(Figure 29.35). This ADP-ribosylation of a single side chain of EF2 blocks its capacity to carry out translocation of the
growing polypeptide chain. Protein synthesis ceases, accounting for the remarkable toxicity of diphtheria toxin.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.5. Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation
Figure 29.33. Eukaryotic Translation Initiation. In eukaryotes, translation initiation starts with the assembly of a
complex on the 5 cap that includes the 40S subunit and Met-tRNAi. Driven by ATP hydrolysis, this complex scans the
mRNA until the first AUG is reached. The 60S subunit is then added to form the 80S initiation complex.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.5. Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation
Table 29.4. Antibiotic inhibitors of protein synthesis
Antibiotic
Action
Streptomycin and other aminoglycosides Inhibit initiation and cause misreading of mRNA (prokaryotes)
Tetracycline
Binds to the 30S subunit and inhibits binding of aminoacyl-tRNAs
(prokaryotes)
Chloramphenicol
Inhibits the peptidyl transferase activity of the 50S ribosomal subunit
(prokaryotes)
Cycloheximide
Inhibits the peptidyl transferase activity of the 60S ribosomal subunit
(eukaryotes)
Erythromycin
Binds to the 50S subunit and inhibits translocation (prokaryotes)
Puromycin
Causes premature chain termination by acting as an analog of aminoacyltRNA (prokaryotes and eukaryotes)
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.5. Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation
Figure 29.34. Antibiotic Action of Puromycin. Puromycin resembles the aminoacyl terminus of an aminoacyl-tRNA.
Its amino group joins the carbonyl group of the growing polypeptide chain to form an adduct that dissociates from the
ribosome. This adduct is stable because puromycin has an amide (shown in red) rather than an ester linkage.
III. Synthesizing the Molecules of Life
29. Protein Synthesis
29.5. Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation
Figure 29.35. Blocking of Translocation by Diphtheria Toxin. Diphtheria toxin blocks protein synthesis in eukaryotes
by catalyzing the transfer of an ADP-ribose unit from NAD+ to diphthamide, a modified amino acid residue in
elongation factor 2 (translocase). Diphthamide is formed by a posttranslational modification (blue) of a histidine residue.