KEY CONCEPTS:
- 16S rRNA plays an active role in the functions of the 30S subunit. It interacts directly with mRNA, with the 50S subunit, and with the anticodons of tRNAs in the P and A sites.
The ribosome was originally viewed as a collection of
proteins with various catalytic activities, held together by protein-protein
interactions and by binding to rRNA. But the discovery of RNA molecules with
catalytic activities (see 24 RNA
splicing and processing) immediately suggests that rRNA might play a more
active role in ribosome function. There is now evidence that rRNA interacts with
mRNA or tRNA at each stage of translation, and that the proteins are necessary
to maintain the rRNA in a structure in which it can perform the catalytic
functions. Several interactions involve specific regions of rRNA:
- The 3
terminus of the rRNA interacts directly with mRNA at initiation.
- Specific regions of 16S rRNA interact directly with the anticodon regions of tRNAs in both the A site and the P site. Similarly, 23S rRNA interacts with the CCA terminus of peptidyl-tRNA in both the P site and A site.
- Subunit interaction involves interactions between 16S and 23S rRNAs (see 6.16 Ribosomal RNA pervades both ribosomal subunits).
Much information about the individual steps of bacterial
protein synthesis has been obtained by using antibiotics that inhibit the
process at particular stages. The target for the antibiotic can be identified by
the component in which resistant mutations occur. Some antibiotics act on
individual ribosomal proteins, but several act on rRNA, which suggests that the
rRNA is involved with many or even all of the functions of the ribosome.
The functions of rRNA have been investigated by two types of
approach. Structural studies show that particular regions of rRNA are located in
important sites of the ribosome, and that chemical modifications of these bases
impede particular ribosomal functions. And mutations identify bases in rRNA that
are required for particular ribosomal functions. Figure
6.45 summarizes the sites in 16S rRNA that have been identified by these
means (for review see Noller, 1991).
An indication of the importance of the 3![]()
end of 16S rRNA is given by its
susceptibility to the lethal agent colicin E3. Produced by some bacteria, the
colicin cleaves ~50 nucleotides from the 3![]()
end of the 16S rRNA of E. coli. The cleavage
entirely abolishes initiation of protein synthesis. Several important functions
require the region that is cleaved: binding the factor IF-3; recognition of
mRNA; and binding of tRNA.
The 3![]()
end
of the 16S rRNA is directly involved in the initiation reaction by pairing with
the Shine-Dalgarno sequence in the ribosome-binding site of mRNA (see Figure 6.16 and Great
Experiments: rRNA-mRNA base pairing selects translational initiator regions in
bacteria). Another direct role for the 3![]()
end of 16S rRNA in protein synthesis is shown by the
properties of kasugamycin-resistant mutants, which lack certain modifications in
16S rRNA. Kasugamycin blocks initiation of protein synthesis. Resistant mutants
of the type ksgA lack a methylase enzyme that introduces four methyl
groups into two adjacent adenines at a site near the 3![]()
terminus of the 16S rRNA. The methylation generates the
highly conserved sequence G–m26A–m26A, found in both prokaryotic
and eukaryotic small rRNA. The methylated sequence is involved in the joining of
the 30S and 50S subunits, which in turn is connected also with the retention of
initiator tRNA in the complete ribosome. Kasugamycin causes
fMet-tRNAf to be released from the sensitive (methylated) ribosomes,
but the resistant ribosomes are able to retain the initiator.
Changes in the structure of 16S rRNA occur when ribosomes
are engaged in protein synthesis, as seen by protection of particular bases
against chemical attack. The individual sites fall into a few groups,
concentrated in the 3![]()
minor
and central domains. Although the locations are dispersed in the linear sequence
of 16S rRNA, it seems likely that base positions involved in the same function
are actually close together in the tertiary structure.
Some of the changes in 16S rRNA are triggered by joining
with 50S subunits, binding of mRNA, or binding of tRNA. They indicate that these
events are associated with changes in ribosome conformation that affect the
exposure of rRNA. They do not necessarily indicate direct participation of rRNA
in these functions. One change that occurs during protein synthesis is shown in
Figure 6.46; it involves a local movement to change the
nature of a short duplex sequence.
The 16S rRNA is involved in both A site and P site function,
and significant changes in its structure occur when these sites are occupied (Moazed and Noller, 1986). Certain distinct regions
are protected by tRNA bound in the A site (see Figure
6.45). One is the 530 loop (which is also is the site of a mutation that
prevents termination at the UAA, UAG, and UGA codons). The other is the
1400-1500 region (so-called because bases 1399-1492 and the adenines at
1492-1493 are two single-stranded stretches that are connected by a long
hairpin). All of the effects that tRNA binding has on 16S rRNA can be produced
by the isolated oligonucleotide of the anticodon stem-loop, so that tRNA-30S
subunit binding must involve this region (Lodmell and Dahlberg, 1997).
The adenines at 1492-1493 provide a mechanism for detecting
properly paired codon-anticodon complexes (Yoshizawa, Fourmy, and Puglisi, 1999). The principle
of the interaction is that the structure of the 16S rRNA responds to the
structure of the first two bases pairs in the minor groove of the duplex formed
by the codon-anticodon interaction. Modification of the N1 position of either
base 1492 or 1493 in rRNA prevents tRNA from binding in the A site. However,
mutations at 1492 or 1493 can be suppressed by the introduction of fluorine at
the 2
A variety of bases in different positions of 16S rRNA are
protected by tRNA in the P site; probably the bases lie near one another in the
tertiary structure. In fact, there are more contacts with tRNA when it is in the
P site than when it is in the A site. This may be responsible for the increased
stability of peptidyl-tRNA compared with aminoacyl-tRNA. This makes sense,
because once the tRNA has reached the P site, the ribosome has decided that it
is correctly bound, whereas in the A site, the assessment of binding is being
made. The 1400 region can be directly cross-linked to peptidyl-tRNA, which
suggests that this region is a structural component of the P site.
The basic conclusion to be drawn from these results is that
rRNA has many interactions with both tRNA and mRNA, and that these interactions
recur in each cycle of peptide bond formation.