A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

Departments of ^*Pediatrics and ^§Biological Chemistry, School of Medicine, University of California, Davis, CA 95616; and ^‡CLONTECH Laboratories, Palo Alto, CA 94303

Communicated by Harry F. Noller, University of California, Santa Cruz, Santa Cruz, CA (received for review December 8, 1997)

Abstract

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (“tail”) domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (“heads”) that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Footnotes

↵ † To whom reprint requests should be addressed at: Department of Pediatrics, School of Medicine, University of California, Davis, Davis, CA 95616. e-mail: avoleinikov{at}ucdavis.edu.
ABBREVIATIONS:

NTF,

N-terminal fragment construct of residues 1–52 of L7/L12;

CTF,

C-terminal fragment construct of residues 53–120 of L7/L12;

TNB,

thionitrobenzoate