Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 116

Chapter 7
Rapid Kinetic Analysis of Protein Synthesis

Marina V. Rodnina and Wolfgang Wintermeyer

7.1 Introduction

Protein synthesis comprises a sequence of consecutive reactions that entail recognition
of substrates by the ribosome, synthesis of chemical bonds, and release of the reac-
tion products. Each of the translation phases—initiation, elongation, termination,
and ribosome recycling—is itself a multistep process. To achieve the overall high
rate of protein synthesis, with ribosomes initiating roughly every 3 s on an mRNA
and about 0.1 s required for each cycle of nascent peptide elongation, each of the
individual reactions on the ribosome must be rapid. This implies that the reaction
intermediates of protein synthesis are short-lived, with lifetimes in the few millisec-
onds range. The overall process is strongly biased towards forward reactions due to
the combination of dynamic fluctuations with irreversible steps of GTP hydrolysis
and peptide bond formation. To study such reactions, transient kinetic techniques
are particularly suitable, as they can reveal sequences of interactions and allow
monitoring the formation and consumption of the reaction intermediates of protein
synthesis in real time.
Rapid kinetic analysis has been utilized to study essentially every step of pro-
teins synthesis. In the first part of this review, we consider the basics of transient
kinetic techniques. The second part gives an overview of two cases where the results
of rapid kinetic studies provided essential insights into the mechanisms of partial
reactions of protein synthesis. We will describe how rapid kinetic analyses contrib-
uted to understanding the mechanisms of chemical reactions catalyzed by the active
site on the 50S ribosomal subunit, i.e., peptide bond formation and peptidyl-tRNA
hydrolysis, during the elongation and termination phases of protein synthesis,