ROLE OF RESIDUE GLU152 IN THE DISCRIMINATION BETWEEN TRANSFER-RNAS BY TYROSYL-TRANSFER RNA-SYNTHETASE FROM BACILLUS-STEAROTHERMOPHILUS

被引:30
作者
VIDALCROS, A [1 ]
BEDOUELLE, H [1 ]
机构
[1] INST PASTEUR,UNITE BIOCHIM CELLULAIRE,CNRS,URA 1129,28 RUE DOCTEUR ROUX,F-75724 PARIS 15,FRANCE
关键词
AMINOACYL-TRANSFER RNA SYNTHETASE; TRANSFER RNA IDENTITY; TRANSFER RNATYR; PROTEIN NUCLEIC ACID RECOGNITION; PROTEIN TOXICITY;
D O I
10.1016/0022-2836(92)90991-R
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Residue Glu152 of tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is close to phosphate groups 73 and 74 of tRNATyr in the structural model of their complex. TyrTS(E152A), a mutant synthetase carrying the change of Glu152 to Ala, was toxic when overproduced in Escherichia coli. The toxicity strongly increased with the growth temperature. It was measured by the ratios of the efficiencies with which the producing cells plated in induced or repressed conditions and at 30 °C or 37 °C. TyrTS(E152Q), TyrTS(E152D) and the wild-type synthetase were not toxic in conditions where TyrTS(E152A) was toxic. The toxicity of TyrTS(E152A) was abolished by additional mutations of the synthetase that prevent the binding of tRNATyr but not by a mutation that prevents the formation of Tyr-AMP. Because TyrTS(E152A) was active for the aminoacylation of tRNATyr, its toxicity could only be due to faulty interactions with non-cognate tRNAs, either their non-productive binding or their mischarging with tyrosine. TyrTS(E152A) and TyrTS(E152Q) mischarged tRNAPhe and tRNAValin vitro with tyrosine unlike TyrTS(E152D) or the wild-type enzyme. Thus, several features of the side-chain in position 152 of TyrTS, including its negative charge, are important for the rejection of non-cognate tRNAs. TyrTS(E152A), TyrTS(E152D) and TyrTS(E152Q) had similar steady-state kinetics parameters for the charging of tRNATyr with tyrosine in vitro, with kcat KM ratios improved 2.5 times relative to the wild-type synthetase. We conclude that the side-chain of residue Glu152 weakens the binding of TyrTS to tRNATyr and prevents its interaction with non-cognate tRNAs. © 1992.
引用
收藏
页码:801 / 810
页数:10
相关论文
共 49 条
[1]   A PROTEIN REQUIRED FOR SPLICING GROUP-I INTRONS IN NEUROSPORA MITOCHONDRIA IS MITOCHONDRIAL TYROSYL-TRANSFER RNA-SYNTHETASE OR A DERIVATIVE THEREOF [J].
AKINS, RA ;
LAMBOWITZ, AM .
CELL, 1987, 50 (03) :331-345
[2]   CLONING AND AMPLIFIED EXPRESSION OF THE TYROSYL-TRANSFER RNA-SYNTHETASE GENES OF BACILLUS-STEAROTHERMOPHILUS AND ESCHERICHIA-COLI [J].
BARKER, DG .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1982, 125 (02) :357-360
[3]   THE TYROSYL-TRANSFER RNA-SYNTHETASE FROM ESCHERICHIA-COLI - COMPLETE NUCLEOTIDE-SEQUENCE OF THE STRUCTURAL GENE [J].
BARKER, DG ;
BRUTON, CJ ;
WINTER, G .
FEBS LETTERS, 1982, 150 (02) :419-423
[4]   A MODEL OF SYNTHETASE TRANSFER-RNA INTERACTION AS DEDUCED BY PROTEIN ENGINEERING [J].
BEDOUELLE, H ;
WINTER, G .
NATURE, 1986, 320 (6060) :371-373
[5]   RECOGNITION OF TRANSFER RNATYR BY TYROSYL-TRANSFER RNA-SYNTHETASE [J].
BEDOUELLE, H .
BIOCHIMIE, 1990, 72 (08) :589-598
[6]   PRODUCTION IN ESCHERICHIA-COLI AND ONE-STEP PURIFICATION OF BIFUNCTIONAL HYBRID PROTEINS WHICH BIND MALTOSE - EXPORT OF THE KLENOW POLYMERASE INTO THE PERIPLASMIC SPACE [J].
BEDOUELLE, H ;
DUPLAY, P .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1988, 171 (03) :541-549
[7]   OVERPRODUCTION OF TYROSYL-TRANSFER RNA-SYNTHETASE IS TOXIC TO ESCHERICHIA-COLI - A GENETIC-ANALYSIS [J].
BEDOUELLE, H ;
GUEZ, V ;
VIDALCROS, A ;
HERMANN, M .
JOURNAL OF BACTERIOLOGY, 1990, 172 (07) :3940-3945
[8]  
BOLLUM FJ, 1959, J BIOL CHEM, V234, P2733
[9]   INTERPRETATION OF INCOMPLETE REACTIONS IN TRANSFER-RNA AMINOACYLATION - AMINOACYLATION OF YEAST TRANSFER RNA-VAL/II WITH YEAST VALYL TRANSFER-RNA SYNTHETASE [J].
BONNET, J ;
EBEL, JP .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1972, 31 (02) :335-&
[10]   STRUCTURE OF TYROSYL TRANSFER-RNA SYNTHETASE REFINED AT 2.3-A RESOLUTION - INTERACTION OF THE ENZYME WITH THE TYROSYL ADENYLATE INTERMEDIATE [J].
BRICK, P ;
BHAT, TN ;
BLOW, DM .
JOURNAL OF MOLECULAR BIOLOGY, 1989, 208 (01) :83-98