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Encyclopedia of Physical Science and Technology EN013D-617 July 27, 2001 11:42

238 Protein Synthesis

between cognate and noncognate tRNAs, while the P-site mutations that favored the 888 conformation were hyper-
must grip the peptidyl–tRNA to prevent dissociation from sensitive to spectinomycin, an antibiotic known to inhibit
the ribosome. EF-G-dependent translocation.

F. Dynamics of Translation
XII. INHIBITORS OF PROTEIN SYNTHESIS
Although the increasing resolution of cryo-EM and crys-
tallographic structures promises molecular level informa- Many compounds have been identified which inhibit dif-
tion in the near future, these structures are snapshots of a ferent steps in protein synthesis, either by mimicking
dynamic particle. During each round of elongation, tRNA translational substrates, blocking movement within the ri-
molecules move through three binding sites on the two bosome, or otherwise interfering with essential interac-
ribosomal subunits, and the mRNA shifts by exactly one tions (Table III). Some of these inhibitors are clinically
codon. While these movements are catalyzed in vivo by relevant antibiotics and others have facilitated functional
EF-Tu and EF-G and depend on GTP hydrolysis, they can studies of the ribosome as described earlier. For example
also be observed in vitro in the absence of elongation fac- aminoglycosides such as streptomycin and gentamycin
tors. Whatever drives the molecular motor therefore must interfere with the decoding center on the 30S subunit. At
be intrinsic to the ribosome. In fact biochemical and ge- low concentrations these antibiotics affect translational
netic studies are beginning to identify switch mechanisms accuracy, while at increased doses they prevent formation
that may drive tRNA selection and translocation. of the 30S initiation complex, leading to cell death. De-
For example, the 16S nucleotides 885–890 and 910– spite the increasing incidence of antibiotic resistance, the
912 can base pair in two different conformations (Fig. 12). aminoglycosides remain useful therapeutic agents against
The 910–912 CUC can form canonical Watson–Crick base gram-negative bacteria.
pairs with the GAG of 888–890 or can pair with the GGG Puromycin is an antibiotic that mimics tyrosyl–or
of 885–887 to give a central G–U wobble pair. Substi- phenylalanyl–tRNA and binds in the ribosomal A-site
tutions demonstrated that both pairings are essential for (Fig. 13). The ribosome will even use puromycin as a sub-
biological activity, suggesting that 16S rRNA switches strate in one round of elongation, forming a peptide bond
between two conformations at different steps in protein between the antibiotic and the P-site peptidyl–tRNA.
synthesis. Furthermore, mutations that favored the “888 This transpeptidation step terminates synthesis of the
conformation” led to restrictive (hyper-accurate) riboso- polypeptide and releases the abortive peptidyl–puromycin
mal phenotypes, while those that stabilized the “885 pair- product.
ing” produced error-prone phenotypes. Structurally, this
triplet-switch-sequence has been located in the 70S crys-
tal structure near the long 16S helix that is part of the XIII. POST-TRANSLATIONAL
decoding region of the small subunit. The switch helix is MODIFICATIONS
certainly involved in tRNA selection, and may also play a
role in translocation, as suggested by the observation that Many proteins, in order to perform their role in the cell,
must be further processed after they are synthesized on the
ribosome. These post-translational modifications involve

TABLE III Inhibitors of Protein Synthesis
Antibiotic Activity

Chloramphenicol Blocks peptidyl transferase function
Erythromycin Blocks translocation of peptidyl–tRNA
Gentamycin Prevents formation of 30S initiation complex
Kirromycin Blocks release of EF-Tu:GDP from ribosome
FIGURE 12 The 16S triplet switch. Genetic studies revealed that
Pseudomonic acid Inhibits isoleucyl–tRNA synthetase
the 885–912 regions of 16S rRNA alternate between base-pairing
conformations, as shown; both are required for cell viability. Mu- Puromycin Mimics tyrosyl–tRNA, terminates elongation
tations that favor the 888 conformation produce ribosomes that Spectinomycin Inhibits EF-G function
are hyper-accurate in protein synthesis, while substitutions that Streptomycin Prevents transition from initiation to elongation
stabilize the 885 conformation lead to error-prone phenotypes. It
Tetracycline Inhibits binding of aminoacyl–tRNAs
is likely that this switch is involved in regulating tRNA selection
to small subunit
and translocation.