Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes.
In previous work we showed that on photolysis of Escherichia coli ribosomes in the presence of [3H]tetracycline (TC) the major protein labeled is S7, and we presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC [Goldman, R. A., Hasan, T., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1983) Biochemistry 22, 359-368]. In this work we use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles we obtain parallel very closely those measured earlier [Nomura, M., Mizushima, S., Ozaki, M., Traub, P., & Lowry, C. V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49-61], with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1396, in one of the major domains of the 30S subunit.
Buck
M A
MA
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Cooperman
B S
BS
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002498:Centrifugation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism*
D001426:Bacterial Proteins; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; D013752:Tetracycline