The proximity of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 determined by cysteine site-directed mutagenesis and protein-protein cross-linking.
Oligonucleotide-directed mutagenesis was used to produce a serine 89 to cysteine 89 substitution in the C-terminal globular domain of Escherichia coli ribosomal protein L7/L12. Cys-89 represented the only cysteine residue in the protein. L7/L12Cys89 was overproduced in E. coli and purified. An allele replacement strain was also constructed. Growth of this strain was indistinguishable from that of wild type. Ribosomes from the allele replacement strain were used to determine the location of the C-terminal domains of L7/L12 by disulfide cross-linking. A new homobifunctional cysteine-specific cross-linking reagent, 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, and diagonal gel electrophoresis were used to identify ribosomal proteins cross-linked to L7/L12Cys89. A cross-link between L7/L12 and the single cysteine in L10 was found, in addition to L7/L12 dimers. The L7/L12Cys89-L10 cross-link locates the C-terminal domain of at least one L7/L12 dimer on the body of the large subunit and supports our previous model (Olson, H. M., Sommer, A., Tewari, D. S., Traut, R. R., and Glitz, D. G. (1986) J. Biol. Chem. 261, 6924-6932) that depicts one of the two dimers of L7/L12 on the surface of the body of the 50 S subunit in a bent conformation with the C-terminal domain in close proximity to the N-terminal domain at the base of the stalk.
Zecherle
G N
GN
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Oleinikov
A
A
Traut
R R
RR
D000483:Alleles; D003432:Cross-Linking Reagents; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D004228:Dithionitrobenzoic Acid; D003545:Cysteine