A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components.
In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 1397-1405 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 1397-1405 lie within a single-stranded sequence that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S subunits, the photolabile oligodeoxyribonucleotide probe site-specifically incorporates into proteins S1, S7, S18, and S21 (identified by SDS-PAGE, RP-HPLC, and antibody affinity chromatography) and into three separate 16S rRNA regions, specifically, nucleotides A-1396, G-1405-A-1408, and A-1492 and A-1493. These results provide clear evidence that G-1405 in 16S rRNA is within 24 A (the distance between G-1405 and the photogenerated nitrene) of proteins S1, S7, S18, and S21 and each of the other nucleotides mentioned above, consistent with other studies of 30S internal structure. Although the probe binds to inactive 30S subunits about as well as to activated 30S subunits, photolysis of the inactive 30S.probe complex leads to a very different pattern of protein labeling, providing strong evidence, at the protein level, that the inactive to activated transition is accompanied by conformational change in the 1400 region of 16S rRNA.
Muralikrishna
P
P
Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323.
Cooperman
B S
BS
D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000378:metabolism; D010782:Photolysis; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*
D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S