Photoaffinity labeling of 30S-subunit proteins S7 and S11 by 4-thiouridine-substituted tRNA(Phe) situated at the P site of Escherichia coli ribosomes

• Published in: RNA (Cambridge) 1997-09, Vol.3 (9), p.1028-1036
• Main Authors: Rosen, K V, Zimmerman, R A
• Format: Article
• Language: English
• Subjects: Affinity Labels - chemistry; Affinity Labels - metabolism; Base Sequence; Binding Sites; Cross-Linking Reagents; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Endoribonucleases - metabolism; Escherichia coli - genetics; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Photochemistry - methods; Ribonuclease P; Ribosomal Proteins - chemistry; Ribosomal Proteins - metabolism; Ribosomes - metabolism; RNA, Catalytic - metabolism; RNA, Transfer, Phe - chemistry; RNA, Transfer, Phe - metabolism; Thiouridine - chemistry; Thiouridine - metabolism; Transcription, Genetic; Viral Proteins
• ISSN: 1355-8382; 1469-9001

4-Thiouridine, a photoreactive analogue of uridine, was randomly incorporated into yeast tRNA(Phe) precursor molecules by transcription with T7 RNA polymerase and the resulting transcripts were converted into mature tRNA(Phe) by treatment with RNase P RNA. The photoreactive tRNA(Phe) was aminoacylated and bound to the P site of Escherichia coli 70S ribosomes in the presence of a poly(U) template. Irradiation of the complexes with light of 300 nm resulted in the covalent crosslinking of nt U20 in the D loop of the tRNA to protein S11 of the 30S ribosomal subunit, whereas nt U33 in the anticodon loop crosslinked to 30S-subunit protein S7. These results allowed us to map the D loop of P site-bound tRNA to the platform of the 30S ribosomal subunit and provided additional information about contacts between protein S7 and the anticodon loop in the cleft between the platform and the subunit head.