278 Chapter 7
of the ribosome, the high-resolution structures have provided a wealth of information about RNA tertiary
architecture and they have provided new insights into the chemical mechanism of the peptidyl transfer
reaction (see below).5,28,29
During translation initiation, a specialized methionyl tRNA (N-formyl methionyl tRNA, or fMet) binds
to the AUG initiation codon, together with the 30S subunit and various initiation factors. This initiation
complexthen binds the 50S subunit, thereby forming an active ribosome. Finally, the EF-Tu shuttle protein
brings in charged tRNA molecules, and the process of translation commences.
The assembled 70S ribosome contains three binding sites for tRNA: The A site, where incoming
aminoacylated tRNA molecules are delivered by EF-Tu; the P-site, which contains tRNA that is bound to
the nascent peptide chain; and the E-site, from which uncharged tRNA molecules exit. Similarly, the
mRNA traverses and exits through a tunnelin the ribosome that helps position the codons during translation.
In order to contribute to the peptide chain, each tRNA must translocatethrough the ribosome, visiting the
A-site, P-site, and E-sites in turn. Translocation is a dynamic, directional process that is facilitated by motor
protein EF-G, which is a tRNA mimic that helps push tRNA from the A-site to the P-site. The process of
translocation is best described by the hybrid states model(Figure 7.30), which has been confirmed and
elaborated by crystallographic analysis of intact, tRNA-bound ribosomes.^57
The multi-step peptidyl transfer reaction is catalyzed within the 50S subunit. During early stages of the
reaction, the incoming amine nucleophile is deprotonated and attacks the activated ester that connects the
nascent peptide chain with the P-site tRNA (Figure 7.31). The resultant tetrahedral intermediate then col-
lapses, resulting in deacylated tRNA in the P-site and an expanded peptide chain on the A-site tRNA.^58
High-resolution structural analysis of the 50S subunit has revealed that the active site for peptidyl transfer
is strikingly devoid of ribosomal proteins, thereby confirming the long-held view that the ribosome is a
ribozyme.^5 While the mechanism of catalysis for peptidyl transfer is still being explored, it is clear that the
major functional groups in the active site are nucleotide bases and backbone moieties.
While there are major differences between prokaryotic and eukaryotic translation, much of the riboso-
mal apparatus is quite similar (Figure 7.27). Bacterial ribosomes jump onto nascent RNA transcripts and
begin making protein even as the mRNA is being transcribed. By contrast, eukaryotic ribosomes utilize
Figure 7.30 The hybrid states model for translocation through the ribosome. A charged tRNA (stick with a circle on
top) moves sequentially through the A, P, and E sites, respectively, during translocation through the
ribosome. According to this model (which has now been confirmed by crystallographic studies), there
are “hybrid states” during each stage of translocation, in which a given tRNA is half-way in one site
(i.e., the anticodon in the A site) and halfway in another (i.e., the acceptor end in the P site). During
translocation, the nascent peptide (a wavy line) is transferred to the amino acid of the incoming tRNA
(aa). The tRNAs shift after translocation and release uncharged tRNA (-OH) from the E site