Bases in 16S rRNA important for subunit association, tRNA binding, and translocation.

Ribosomes contain three tRNA binding sites: the aminoacyl site (A site), the peptidyl site (P site), and the exit site (E site). During protein synthesis, A and P site tRNAs move in a precise and coordinated manner to the P and E sites, respectively. Early biochemical and biophysical studies showed that the movement of the tRNAs occurs in two steps (1, 2). First, following peptide bond formation, the acceptor ends of deacylated and peptidyl tRNAs translocate spontaneously into the 50S subunit's E and P sites, respectively. The anticodon ends of deacylated and peptidyl tRNAs maintain their interactions with the 30S subunit's P and A sites, respectively, resulting in hybrid P/E and A/P binding states. Second, EF-G binds to the ribosome and hydrolyzes GTP. GTP hydrolysis accelerates the translocation of the anticodon end of the tRNAs and the associated mRNA by one codon relative to the 30S subunit (3). Recent experiments are consistent with a step wise movement of the tRNAs through the ribosome (4-10). Strikingly, during translocation, the ribosome goes through large-scale conformational changes as shown by cryo-EM reconstruction of stalled translocation intermediates (11-14). The changes depicted by cryo-EM showed the large and small ribosomal subunits undergoing a ratchet like movement relative to each other during translocation. This indicates that translocation involves multiple conformational changes in both subunits. However, conformational changes in the ribosome are not well understood and the molecular basis of translocation is not yet clear. Numerous studies suggest that the rRNAs play an important role during translocation. First, mutations or modifications in rRNAs confer resistance to antibiotics, such as viomycin, thiostrepton, and spectinomycin, that block specific steps in translocation (15-17). Most of these antibiotics protect nucleotides in rRNAs from chemical probes indicating that they bind directly to the rRNAs (18, 19). Indeed, this has been confirmed by recent x-ray crystallographic studies, which showed that antibiotics that inhibit translocation, such as thiostrepton, spectinomycin and hygromycin B, mainly contact the rRNAs (20-23). Second, EF-G protects nucleotides in 23S rRNA and has been cross-linked to 23S rRNA suggesting that contacts with the rRNAs are critical for translocation (24, 25). Third, disruption of specific interactions between the tRNAs and the rRNAs inhibit translocation (7, 26-29). Even though high-resolution structures of the ribosome are now available, it is difficult to predict which bases in the rRNAs are important for specific steps in translation. This gap in understanding the functional significance of the structure needs to be filled by biochemical and biophysical studies that analyze interesting features revealed by the structures. However, systematically interrogating individual bases for their functional role in translation can become tedious and expensive. On the other hand, modification-interference methods allow the simultaneous analysis of many residues in one experiment, and have been used for identifying functionally important nucleotides in a variety of RNA structures, RNA-protein complexes and catalytic RNAs. Specifically, modification-interference methods have proven useful for identifying bases in rRNA that are essential for tRNA binding to the ribosomal A and P sites (30-32). Here, we describe a powerful in vitro modification-interference method for identifying bases in 16S rRNA that are critical for translocation. The modification-interference method is based on the site-specific cross-linking of tRNA to 16S rRNA in ribosomes that have undergone translocation. Using this modification-interference approach, eleven bases were identified that may play an important role during translocation. Interestingly, five of these bases have never been implicated as being important for translation. These five bases were mutated in the 16S rRNA and the 16S rRNA mutants were systematically analyzed to examine their role in translation. Our results show that these bases located remotely from the tRNA binding sites affect subunit association, tRNA binding to the P site, and translocation of mRNA-tRNA complex by the ribosome.