Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

Abstract

The coupled movement of tRNA–mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site–bound tRNA^Met with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P- and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site–bound tRNA^Met to investigate the contributions of single 2′-hydroxyl groups in the 5′ strand of the acceptor arm for translocation into the ribosomal E-site. EF-G–dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site–bound tRNA^Met between positions 1–17 play a critical role during translocation. This suggests that either these 2′-hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

Keywords

• ribosome
• translocation
• tRNA
• mRNA
• EF-G
• mechanism