An Investigation of the Conformational Properties of Ribosomes Using N‐Ethylmalemide as a Probe

Abstract

The reactivity of ribosomal proteins towards N-ethylmaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichiu coli. The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation. The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity. The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed.

Photoincorporation of tetracycline into Escherichia coli ribosomes. Identification of the major proteins photolabeled by native tetracycline and tetracycline photoproducts and implications for the inhibitory action of tetracycline on protein synthesis

“…As may be seen, the only major area of radioactivity is found near the group S3, S4, S5, and S7. This result is not inconsistent with the one-dimensional NaDodSO,-urea-polyacrylamide gel electrophoresis results because retarded migration of affinity labeled proteins in two-dimensional polyacrylamide gel electrophoresis analysis has been noted earlier (Czernilofsky et al, 1974;Grant et al, 1979;Goldman et al, 1980). However, from Figure 4 it is clear that protein S8 and the area around it contain little radioactivity.…”

“…TC and [7-3H]TC (1 Ci/mmol) were obtained and utilized as previously described (Goldman et al, 1980). E-TC was obtained as a mixture with TC by incubation of TC in buffer A for 16 h at 25 "C as previously described (McCormick et al, 1957).…”

“…[ 14C]Phe-tRNAPhc binding to ribosomes was measured with a filter assay as described previously (Goldman et al, 1980). Reaction mixtures were 1% in Me2S0 as a precaution to fully solubilize T C photoproducts.…”

“…Such placement of S7 suggests a structural model for the mechanism of tetracycline inhibition of aminoacyl-tRNA binding to the A site in which tetracycline binding prevents simultaneous two-site attachment of aminoacyl-tRNA at both the codonanticodon site and the peptidyltransferase center. (Goldman et al, 1980) and the rat liver ribosome (Reboud et al, 1982). In our previous publication (Goldman et al, 1980) we demonstrated that TC, an inhibitor of tRNA binding to the A site, will photoincorporate into E .…”

“…(Goldman et al, 1980) and the rat liver ribosome (Reboud et al, 1982). In our previous publication (Goldman et al, 1980) we demonstrated that TC, an inhibitor of tRNA binding to the A site, will photoincorporate into E . coli ribosome and identified the major proteins labeled as S18 and S4, and, to a lesser extent, S7, S13, and S14XGoldman et al , 1980).…”