INTERACTION OF ESCHERICHIA-COLI TRANSFER RNASER WITH ITS COGNATE AMINOACYL-TRANSFER RNA-SYNTHETASE AS DETERMINED BY FOOTPRINTING WITH PHOSPHOROTHIOATE-CONTAINING TRANSFER-RNA TRANSCRIPTS

被引:108
作者
SCHATZ, D
LEBERMAN, R
ECKSTEIN, F
机构
[1] MAX PLANCK INST EXPTL MED,CHEM ABT,HERMANN REIN STR 3,W-3400 GOTTINGEN,GERMANY
[2] EUROPEAN MOLEC BIOL LAB,F-38042 GRENOBLE,FRANCE
关键词
PHOSPHOROTHIOATE-TRANSFER RNA; IODINE CLEAVAGE; SEQUENCING;
D O I
10.1073/pnas.88.14.6132
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:6132 / 6136
页数:5
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