15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis.
Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.
Kawai
G
G
Faculty of Engineering, University of Tokyo, Japan.
Takayanagi
M
M
Hayashi
N
N
Niimi
T
T
Sanpei
G
G
Mizobuchi
K
K
Miyazawa
T
T
Tokoyama
S
S
Watanabe
K
K
D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D012329:RNA, Bacterial / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012329:RNA, Bacterial; D012343:RNA, Transfer