Interactions between the acceptor arm of tRNA and the large subunit of the bacterial ribosome important for translocation

A fundamental step during the elongation phase of protein synthesis is the stepwise movement of the tRNAs through the ribosome. The tRNAs bind to the ribosome and make interactions with the large and small ribosomal subunits [1]. The specific contacts made between the tRNA and the ribosomes during the elongation phase have yet to be elucidated.;We determined ribose 2'-hydroxyl groups in the tRNA acceptor arm that are important for translocation. We demonstrated that the ribosome will bind and translocate a tRNA molecule having a "nick" in its TPsiC-loop from the ribosomal P-site to the E-site. Systematic analysis of "nicked" tRNAs having single ribose 2'-hydroxyl modifications in the 3'-acceptor arm of the tRNA identified two 2'-hydroxyl groups, one at position at 71 and another at position 76, that are important for translocation. Crystallographic data showed the 2'-hydroxyl group at positions 71 and 76 contacted the backbone of 23S rRNA residues 1892 and 2433--2434, respectively, in the ribosomal E site. These results establish a set of functional interactions between P-site tRNA and 23S rRNA essential for translocation [2].;In addition we developed a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA through the ribosome. This method uses the fluorescent probe pyrene covalently attached to the 3' end of a short mRNA at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods [3]. The assay was further optimized through the use of HPLC purified aminoacylated tRNAs and native EF-G yielding translocation rates comparable to that observed in vivo.;Utilizing the fluorescence based assay we further investigated interactions between the tRNA acceptor arm and the ribosome that are important for translocation. We examined the ribose 2'-hydroxyl groups in the tRNA 5'-acceptor arm by assaying tRNAs having a "nicked" D-loop. Systematic analysis of the fragmented tRNA having single ribose 2'-hydroxyl in the 5'-acceptor arm did not identify any positions important for translocation. These results are consistent with the available x-ray crystallographic data of the ribosome.