Studies on the mechanisms of translocation and termination

Abstract

This chapter addresses two long-standing questions concerning ribosome structure and function: (i) How are the mRNA and tRNAs moved through the ribosome following formation of each peptide bond? and (ii) how does recognition of a stop codon result in hydrolysis of peptidyl-tRNA? Not surprisingly, results from structural biology have played an important part in formulating mechanistic models for both of these processes. Although structural information is essential for understanding the detailed molecular mechanisms of such processes, it is in itself insufficient for establishing whether or not they are correct. There are already sufficient published examples of false mechanistic inferences based on ribosome structures to remind us that such models need to be tested experimentally, preferably by diverse approaches. Key aspects of the standard models for translocation and termination have emerged from structural observations — cryoEM reconstructions and x-ray crystallography, respectively. Both models have been subjected to experimental tests of various kinds, a process that continues in many laboratories. In the first part of this chapter, we describe the results of experiments using both single-molecule and bulk fluorescence methods to examine the relationship between intersubunit movement, hybrid-states binding of tRNA a6nd translocation. In the second part, we discuss a model for the mechanism of translation termination based on the x-ray crystal structures of the translation termination complexes, and some experimental tests of the model.