Photochemical Cross-Linking of tRNAArg to the 30S Ribosomal Subunit Using Aryl Azide Reagents Attached to the Anticodon Loop†

Jan Kan Chen, Lothar A. Franke, Stephen S. Hixson, Robert A. Zimmermann

Research output: Contribution to journalJournal Article peer-review

12 Scopus citations

Abstract

The 2-thiocytidine residue at position 32 of [formula omitted] from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetyl-arginyl-[formula omitted] derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-[formula omitted] derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.

Original languageEnglish
Pages (from-to)4777-4784
Number of pages8
JournalBiochemistry
Volume24
Issue number18
DOIs
StatePublished - 01 08 1985
Externally publishedYes

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