Open AccessInteraction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.
Author(s) -
Dagmar Schatz,
Reuben Leberman,
Fritz Eckstein
Publication year - 1991
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.88.14.6132
Subject(s) - transfer rna , footprinting , escherichia coli , biology , aminoacyl trna synthetase , microbiology and biotechnology , rna , biochemistry , gene , transcription factor
A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.