Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 117

respectively. To illustrate the power of the stopped-flow technique, we describe
fluorescence experiments that lead to understanding the distinct roles of the elonga-
tion factor G (EF-G) in the catalysis of tRNA-mRNA translocation during elonga-
tion and subunit splitting in the ribosome recycling phase.

7.2 Rapid Kinetic Techniques

Rapid kinetic measurements allow for the detection of transient reaction intermedi-
ates. Following the time courses of the formation of intermediates and their con-
sumption in subsequent reactions provides the sequence of events as well as reaction
rate constants (for a comprehensive overview, see Johnson 2003 ) . Among the rapid
kinetic methods that have been used to study protein synthesis, stopped-flow and
quench-flow techniques are particularly important. In a stopped-flow apparatus, two
reactants are rapidly mixed and a change in an optical parameter is monitored, such
as fluorescence, absorption, or light scattering. Commercially available machines
provide a reliable signal 1 ms after mixing (the dead-time of the apparatus) and
allows to follow reactions with rates of up to 500 s −1 . In quench- fl ow experiments,
which in studies of protein synthesis are usually used to determine the rates of
chemical reactions, reactions are initiated and stopped (quenched) rapidly, and the
products are quantified offline by a variety of analytical methods. The minimum
reaction time of commercial quench-flow machines is close to 3 ms; higher time
resolution can be reached by using the continuous-flow mode, albeit at the expense
of the consumption of large amounts of substrate. Recently, a number of additional
rapid kinetic techniques have been established, such as rapid filtration, time-resolved
cross-linking, or footprinting (Fabbretti et al. 2007 ). Flash photolysis, which has
been successfully utilized in other fields, is another yet underexploited method to
study translation intermediates. In the following, we focus on the standard stopped-
flow and quench-flow setups which are broadly used in ribosome research.
Dissecting a reaction mechanism by means of transient kinetics entails several
steps. The first step is the choice of the technique, stopped-flow or quench flow, and
the reporter groups appropriate to study the particular reaction, for stopped-flow
usually fluorescent groups. The positions of labels should be chosen using structural
information (wherever available) and sequence conservation alignments. Because
the quality of the signal depends critically on the position and the spectroscopic
properties of the fluorescence label, it is advantageous to try different fluorophores
and various positions in proteins/RNA for labeling. The functional properties of the
labeled molecules have to be characterized by biochemical methods. The second
step is to carefully choose the reaction conditions for the stopped-flow or quench-
flow experiments. This step is very important because the reaction conditions, in
particular the concentrations of reactants, determine the complexity of the kinetic
data obtained. Finally, the evaluation of the data should result in a kinetic model that
can be tested by further experiments.