Transfer RNAPhe isoacceptors possess non‐identical set of identity elements at high and low Mg2+ concentration

FEBS Letters

Ksenzenko, Vladimir N²  Kholod, Natalia S²  Pan'kova, Natalia V²  Kisselev, Lev L¹  Mayorov, Sergey G²  Krutilina, Antonina I²  Shlyapnikov, Michael G² 

[1] Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 117984, Russia;Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia

关键词: Protein–nucleic acid recognition;    Aminoacyl-tRNA synthetase;    tRNA gene transcripts;    tRNA identity elements;    bacteriophage T5;    tRNA conformation;    aaRS;    aminoacyl-tRNA synthetase(s);    E.C. 6.1.1;    FRS;    phenylalanyl-tRNA synthetase;    Phe-tRNAPhe;    phenylalanyl-tRNAPhe;    EF–Tu;    elongation factor Tu;    PAGE;    polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(97)00608-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Primary structures of phage T5- and Escherichia coli-encoded tRNA^Phe are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA^Phe gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA^Phe gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg²⁺ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA^Phe mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg²⁺ but not at optimal 4 mM Mg²⁺; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg²⁺ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg²⁺; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg²⁺ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA^Phe recognition by E. coli FRS at low and high Mg²⁺ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

【 授权许可】

Unknown