KEY CONCEPTS:
- Peptidyl transferase activity resides exclusively in the 23S rRNA.
The sites involved in the functions of 23S rRNA are less
well identified than those of 16S rRNA, but the same general pattern is
observed: bases at certain positions affect specific functions. Bases at some
positions in 23S rRNA are affected by the conformation of the A site or P site.
In particular, oligonucleotides derived from the 3![]()
CCA terminus of tRNA protect a set of bases in 23S rRNA
which essentially are the same as those protected by peptidyl-tRNA. This
suggests that the major interaction of 23S rRNA with peptidyl-tRNA in the P site
involves the 3![]()
end of the
tRNA.
The tRNA makes contacts with the 23S rRNA in both the P and
A sites. At the P site, G2552 of 23S rRNA base pairs with C74 of the peptidyl
tRNA. A mutation in the G in the rRNA prevents interaction with tRNA, but
interaction is restored by a compensating mutation in the C of the amino
acceptor end of the tRNA. At the A site, G2553 of the 23S rRNA base pairs with
C75 of the aminoacyl-tRNA. So there is a close role for rRNA in both the
tRNA-binding sites. Indeed, when we have a clearer structural view of the
region, we should be able to understand the movements of tRNA between the A and
P sites in terms of making and breaking contacts with rRNA (Samaha, Green, and Noller, 1995).
Another site that binds tRNA is the E site, which is
localized almost exclusively on the 50S subunit. Bases affected by its
conformation can be identified in 23S rRNA.
What is the nature of the site on the 50S subunit that
provides peptidyl transferase function? The involvement of rRNA was first
indicated because a region of the 23S rRNA is the site of mutations that confer
resistance to antibiotics that inhibit peptidyl transferase.
A long search for ribosomal proteins that might possess the
catalytic activity has been unsuccessful. More recent results suggest that the
ribosomal RNA of the large subunit has the catalytic activity. Extraction of
almost all the protein content of 50S subunits leaves the 23S rRNA associated
largely with fragments of proteins, amounting to <5% of the mass of the ribosomal proteins. This
preparation retains peptidyl transferase activity. Treatments that damage the
RNA abolish the catalytic activity (Noller, Hoffarth, and Zimniak, 1992).
Following from these results, 23S rRNA prepared by
transcription in vitro can catalyze the formation of a peptide bond
between Ac-Phe-tRNA and Phe-tRNA. The yield of Ac-Phe-Phe is very low,
suggesting that the 23S rRNA requires proteins in order to function at a high
efficiency. But since the rRNA has the basic catalytic activity, the role of the
proteins must be indirect, serving to fold the rRNA properly or to present the
substrates to it. The reaction also works, although less effectively, if the
domains of 23S rRNA are synthesized separately and then combined. In fact, some
activity is shown by domain V alone, which has the catalytic center. Activity is
abolished by mutations in position 2252 of domain V that lies in the P
site.
The crystal structure of an archaeal 50S subunit shows that
the peptidyl transferase site basically consists of 23S rRNA (Ban et al., 2000). There is no protein within 18Å of the active site where the transfer reaction occurs
between peptidyl-tRNA and aminoacyl-tRNA!
Peptide bond synthesis requires an attack by the amino group
of one amino acid on the carboxyl group of another amino acid. Catalysis
requires a basic residue to accept the hydrogen atom that is released from the
amino group, as shown in Figure 6.48. If rRNA is the
catalyst, it must provide this residue, but we do not know how this happens. The
purine and pyrimidine bases are not basic at physiological pH. A highly
conserved base (at position 2451 in E. coli) had been implicated, but
appears now neither to have the right properties nor to be crucial for peptidyl
transferase activity (Bayfield et al., 2001; Thompson et al., 2001).
Proteins that are bound to the 23S rRNA outside of the
peptidyl transfer region are almost certainly required to enable the rRNA to
form the proper structure in vivo. The idea that rRNA is the catalytic
component is consistent with the results discussed in 24 RNA splicing and processingthat
identify catalytic properties in RNA that are involved with several RNA
processing reactions. It fits with the notion that the ribosome evolved from
functions originally possessed by RNA.
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