KEY CONCEPTS:
- Interactions involving rRNA are a key part of ribosome function.
- The environment of the tRNA-binding sites is largely determined by rRNA.
The basic message to remember about the ribosome is that it
is a cooperative structure that depends on changes in the relationships among
its active sites during protein synthesis. The active sites are not small,
discrete regions like the active centers of enzymes. They are large regions
whose construction and activities may depend just as much on the rRNA as on the
ribosomal proteins. The crystal structures of the individual subunits and
bacterial ribosomes give us a good impression of the overall organization and
emphasize the role of the rRNA. The most recent structure, at 5.5 Å resolution, clearly identifies the locations of the
tRNAs and the functional sites (Yusupov et al., 2001; for review see Ramakrishnan, 2002). We can now account for many
ribosomal functions in terms of its structure (for review see Lafontaine and Tollervey, 2001, Moore and Steitz, 2003).).
Ribosomal functions are centered around the interaction with
tRNAs. Figure 6.42 shows the 70S ribosome with the
positions of tRNAs in the three binding sites (Cate et al., 1999). The tRNAs in the A and P sites
are nearly parallel to one another. All three tRNAs are aligned with their
anticodon loops bound to the mRNA in the groove on the 30S subunit. The rest of
each tRNA is bound to the 50S subunit. The environment surrounding each tRNA is
mostly provided by rRNA. In each site, the rRNA contacts the tRNA at parts of
the structure that are universally conserved.
It has always been a big puzzle to understand how two bulky
tRNAs can fit next to one another in reading adjacent codons. The crystal
structure shows a 45° kink in the mRNA between the
P and A sites, which allows the tRNAs to fit as shown in the expansion of Figure 6.43. The tRNAs in the P and A sites are angled at
26° relative to each other at their anticodons.
The closest approach between the backbones of the tRNAs occurs at the 3![]()
ends, where they converge to
within 5 Å (perpendicular to the plane of the
screen). This allows the peptide chain to be transferred from the peptidyl-tRNA
in the A site to the aminoacyl-tRNA in the A site.
Aminoacyl-tRNA is inserted into the A site by EF-Tu, and its
pairing with the codon is necessary for EF-Tu to hydrolyze GTP and be released
from the ribosome (see 6.10 Elongation factor Tu loads aminoacyl-tRNA into the A site
). EF-Tu initially places the aminoacyl-tRNA into the small subunit, where the
anticodon pairs with the codon. Movement of the tRNA is required to bring it
fully into the A site, when its 3![]()
end enters the peptidyl transferase center on the large
subunit. There are different models for how this process may occur. One calls
for the entire tRNA to swivel, so that the elbow in the L-shaped structure made
by the D and TΨC arms moves into the ribosome,
enabling the TΨC arm to pair with rRNA (Valle et al., 2002). Another calls for the internal
structure of the tRNA to change, using the anticodon loop as a hinge, with the
rest of the tRNA rotating from a position in which it is stacked on the 3![]()
side of the anticodon loop to one
in which it is stacked on the 5![]()
side (Simonson and Simonson, 2002). Following the
transition, EF-Tu hydrolyzes GTP, allowing peptide synthesis to proceed.
Translocation involves large movements in the positions of
the tRNAs within the ribosome. The anticodon end of tRNA moves ~28 Å from the A site to the P site, and then a further 20
Å from the P site to the E site. Because of the
angle of each tRNA relative to the anticodon, the bulk of the tRNA moves much
larger distances, 40 Å from A to P site, and 55
Å from P site to E site. This suggests that
translocation requires a major reorganization of structure.
For many years, it was thought that translocation could
occur only in the presence of the factor EF-G. However, the antibiotic
sparsomyicn (which inhibits the peptidyl transferase activity) triggers
translocation (Fredrick and Noller, 2003). This suggests that the
energy to drive translocation actually is stored in the ribosome after peptide
bond formation has occurred. Usually EF-G acts on the ribosome to release this
energy and enable it to drive translocation, but sparsomycin can have the same
role. Sparsomycin inhibits peptidyl transferase by binding to the peptidyl-tRNA,
blocking its interaction with aminoacyl-tRNA. It probably creates a conformation
that resembles the usual post-translocation conformation, which in turn promotes
movement of the peptidyl-tRNA. The important point is that translocation is an
intrinsic property of the ribosome.
The hybrid states model suggests that translocation may take
place in two stages, with one ribosomal subunit moving relative to the other to
create an intermediate stage in which there are hybrid tRNA-binding sites (50S
E/30S P and 50S P/ 30S A) (see Figure 6.29). Comparisons
of the ribosome structure between pre- and post-translocation states, and
comparisons in 16S rRNA conformation between free 30S subunits and 70S
ribosomes, suggest that mobility of structure is especially marked in the head
and platform regions of the 30S subunit. An interesting insight on the hybrid
states model is cast by the fact that many bases in rRNA involved in subunit
association are close to bases involved in interacting with tRNA. This suggests
that tRNA-binding sites are close to the interface between subunits, and carries
the implication that changes in subunit interaction could be connected with
movement of tRNA.
Much of the structure of the ribosome is occupied by its
active centers. The schematic view of the ribosomal sites in Figure 6.44 shows they comprise about two thirds of the
ribosomal structure. A tRNA enters the A site, is transferred by translocation
into the P site, and then leaves the (bacterial) ribosome by the E site. The A
and P sites extend across both ribosome subunits; tRNA is paired with mRNA in
the 30S subunit, but peptide transfer takes place in the 50S subunit. The A and
P sites are adjacent, enabling translocation to move the tRNA from one site into
the other. The E site is located near the P site (representing a position en
route to the surface of the 50S subunit). The peptidyl transferase center is
located on the 50S subunit, close to the aminoacyl ends of the tRNAs in the A
and P sites (see 6.18 16S rRNA plays
an active role in protein synthesis).
All of the GTP-binding proteins that function in protein
synthesis (EF-Tu, EF-G, IF-2, RF1,2,3) bind to the same factor-binding site
(sometimes called the GTPase center), which probably triggers their hydrolysis
of GTP. It is located at the base of the stalk of the large subunit, which
consists of the proteins L7/L12. (L7 is a modification of L12, and has an acetyl
group on the N-terminus.) In addition to this region, the complex of protein L11
with a 58 base stretch of 23S rRNA provides the binding site for some
antibiotics that affect GTPase activity. Neither of these ribosomal structures
actually possesses GTPase activity, but they are both necessary for it. The role
of the ribosome is to trigger GTP hydrolysis by factors bound in the
factor-binding site.
Initial binding of 30S subunits to mRNA requires protein S1,
which has a strong affinity for single-stranded nucleic acid. It is responsible
for maintaining the single-stranded state in mRNA that is bound to the 30S
subunit (Sengupta, Agrawal, and Frank, 2001). This action is
necessary to prevent the mRNA from taking up a base-paired conformation that
would be unsuitable for translation. S1 has an extremely elongated structure and
associates with S18 and S21. The three proteins constitute a domain that is
involved in the initial binding of mRNA and in binding initiator tRNA. This
locates the mRNA-binding site in the vicinity of the cleft of the small subunit
(see Figure 6.38). The 3![]()
end of rRNA, which pairs with the mRNA initiation site,
is located in this region.
The initiation factors bind in the same region of the
ribosome. IF-3 can be crosslinked to the 3![]()
end of the rRNA, as well as to several ribosomal
proteins, including those probably involved in binding mRNA. The role of IF-3
could be to stabilize mRNA·30S subunit binding;
then it would be displaced when the 50S subunit joins.
The incorporation of 5S RNA into 50S subunits that are
assembled in vitro depends on the ability of three proteins, L5, L8, and L25, to
form a stoichiometric complex with it. The complex can bind to 23S rRNA,
although none of the isolated components can do so. It lies in the vicinity of
the P and A sites.
A nascent protein debouches through the ribosome, away from
the active sites, into the region in which ribosomes may be attached to
membranes (see 8 Protein
localization). A polypeptide chain emerges from the ribosome through an exit
channel, which leads from the peptidyl transferase site to the surface of the
50S subunit. The tunnel is composed mostly of rRNA. It is quite narrow, only 1-2
nm wide, and ~10 nm long. The nascent polypeptide emerges from the ribosome ~15
Å away from the peptidyl transferase site. The
tunnel can hold ~50 amino acids, and probably constrains the polypeptide chain
so that it cannot fold until it leaves the exit domain.