Affinity Labeling Techniques for Examining Functional Sites of Ribosomes

Abstract

INTRODUCTION
No other technique can provide the detailed picture of macromolecular functional sites than the one potentially available from X-ray diffraction studies. However, a review of past studies on enzymes reveals that active site-directed chemical reagents have been particularly effective for identifying amino acid residues that are important for function. It seems natural to extend these successful techniques to attempt to identify ribosomal components which are located in various functional sites. Here attention will be restricted specifically to affinity labels. These are chemically reactive or activatable analogs of substrates, inhibitors or effectors. If properly designed, they will bind to a functional site in a manner that closely mimics that of the natural ligand. Once bound, the affinity label is capable of reacting covalently with residues of the binding site. Subsequent analysis of the covalent products permits identification of components of this site. The affinity labeling technique is well established. For a summary of the principles and recent applications, see the excellent reviews by Singer (1967), Shaw (1970) and Knowles (1972). In this brief report, we shall concentrate on some of the particular problems encountered when affinity labels are applied to the ribosome. A survey of successful results to date and possible future directions will be given.

Design of Affinity Labels
Two variables must be examined in constructing a useful affinity label: the choice of the reactive moiety and the site of its attachment to a substrate or ligand. With any affinity experiment, the goal is to optimize reaction at the...