Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.
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.
Kholod
N S
NS
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region.
Pan'kova
N V
NV
Mayorov
S G
SG
Krutilina
A I
AI
Shlyapnikov
M G
MG
Kisselev
L L
LL
Ksenzenko
V N
VN
D000215:Acylation; D000926:Anticodon; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013604:T-Phages / Q000235:genetics*
D000926:Anticodon; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D012367:RNA, Viral; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium