Analogs of Lysyl-tRNA as Probes of Ribosome and Elongation Factor Tu Structure and Function

Abstract

tRNA is the substrate in nearly all of the partial reactions of protein biosynthesis. Therefore, the most direct means of investigating the structural and functional states of the ribosome and associated macromolecules is to monitor the tRNA. With this approach, it is also possible to correlate directly a structural state with a particular functional state, as the functional state of a tRNA in a ribosomal complex can be determined by biochemical assay.

Selective observation of the tRNA is complicated by the presence of rRNA and mRNA in the ribosomal complex. Techniques that measure an intrinsic property of RNA are generally unsuitable because a tRNA constitutes only about 1% of the total RNA in the complex. This problem is circumvented by covalently attaching to a tRNA a tag or probe moiety that is easily observable above the background signal of the system. This paper describes our work using a unique class of aminoacyl-tRNA analogs that have probe moieties attached to the side chain of the lysine in lysyl-tRNA.

THE PROBE ATTACHMENT SITE
Probes attached to (or in place of) various unusual bases in the tRNA have provided information about each of the tRNA loop regions and their environments. At the aminoacyl end of the molecule, probes have usually been attached to the α-amino nitrogen of the amino acid. These modified tRNAs are analogs of peptidyl-tRNA, however, and cannot interact with elongation factor Tu (EF-Tu; I will confine my discussion to the Escherichia coli system) because of the blocked α-amino group (Miller...