Page 195 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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

Protein Synthesis 235

AA–tRNA spends more time in complex with the ribo- increase fidelity, apparently by slowing down translation
some, GTP hydrolysis is likely to occur prior to dissoci- to allow more thorough proofreading.
ation of the tRNA, promoting tight binding of this tRNA
in the A-site prior to peptide bond formation. XI. THE RIBOSOME
In addition to passive kinetic proofreading, selection
of the correct codon–anticodon interaction is proposed to
The bacterial ribosome has been the subject of intense
trigger a conformational change in the ribosome that is
study for several decades. Although the general mecha-
transmitted to EF-Tu. Hydrolysis of GTP is then acceler-
nisms of protein synthesis (as outlined earlier) are rea-
ated for the cognate compared to the noncognate substrate.
sonably well understood, only recently have structures
Recentstructuralevidenceshowsthatstructuralrearrange-
emerged which make a molecular description of ribosome
ments do occur upon binding of the cognate AA–tRNA,
function appear possible. Because of the high degree of
suggesting that selection of the correct substrate depends
functional and sequence conservation between bacterial
on an induced fit mechanism. How the codon–anticodon
and eukaryotic components of the ribosome, structural in-
interaction is detected by the ribosome is not currently
formation obtained using bacterial ribosomes is expected
understood.
to contribute to a universal understanding of ribosomal
architecture.
H. Ribosomal Contributions to Accuracy
Naturallyoccurringmutationshavebeenidentifiedinribo- A. Composition of the Ribosome
somal proteins and rRNA that increase or decrease trans-
The E. coli ribosome contains 3 ribosomal RNA (rRNA)
lational accuracy. These have provided clues as to which
molecules and about 50 proteins, divided between two
regions of the ribosome are involved in proofreading, al-
unequal subunits. (The number of proteins is not entirely
though detailed mechanisms are not known. The small
certain, as some proteins are loosely associated with the ri-
subunit, which contains the decoding site, has several pro-
bosome but may not be integral to its function.) The small
teins that are likely involved in controlling missense and
subunit sediments as a 30S particle, which has a single
processivity errors. For example, mutations have been
16S rRNA molecule and 21 proteins, S1–S21 (S in this
identified in proteins S4 and S5 that reduce the level of
case indicates a small subunit protein). The large subunit
translational fidelity (these are called ribosomal ambiguity
has two rRNAs (5S and 23S) and at least 34 proteins, with
mutations). In contrast, S12 mutants increase the accuracy
L-prefixes. In the cell, ribosomal subunits spontaneously
of translation by conferring resistance to streptomycin, an
assemble from their protein and rRNA components and
error-causing antibiotic.
are brought together as a functional complex with mRNA
The small subunit 16S rRNA also contains regions that
and tRNA at the initiation step of translation. Individual
appear to be involved in translational accuracy. In par-
subunits and functional ribosomes can also be reconsti-
ticular, substitutions at nucleotides in the so-called 530
tuted in vitro under the right salt and temperature condi-
loop lead to changes in the error rate of protein synthesis.
tions. The determination of these conditions has greatly
This rRNA loop is known to be spatially near the proteins
facilitated structural studies of the ribosome. For exam-
S4, S5, and S12, and effects of nucleotide changes in the
ple, subunits can be reconstituted in the absence of one or
530 loop parallel those observed for the proteins. Thus,
more proteins and then assayed for structural integrity and
the rRNA and proteins in this part of the small subunit
function. Such experiments determined which proteins are
together act as a proofreading domain. Substitutions at
essential, either because they are needed for subsequent
some of these nucleotides increase the rate of missense
binding of other proteins or because they are necessary for
or frameshift errors, while others are detrimental because
ribosomal function.
theypreventbindingoftheEF-Tu:GTP:AA–tRNAternary
complex. Still other mutations actually increase the accu- B. Sequence Conservation of
racy of translation, in that they make the ribosomes resis- Ribosomal Components
tant to error-inducing antibiotics.
The toxins α-sarcin and ricin inactivate ribosomes by Nucleotide sequences are now known for rRNAs from
altering a highly conserved sequence in 23S-like rRNAs hundreds of organisms. Comparisons of these sequences
(the sarcin–ricin loop). Elongation factors Tu and G bind allowed predictions of the secondary structures of these
near this loop, suggesting that it is involved in AA–tRNA RNAs (which regions are single stranded and which base
selection and translocation. Protein L6 and the sarcin– pair to form helices). Interestingly, the rRNA secondary
ricin loop are involved in translational accuracy control, structures are evolutionarily conserved, while the individ-
as suggested by the mutations in these components that ual nucleotides that make up these structures often are not.