Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation.
The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Yue
D
D
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Kintanar
A
A
Horowitz
J
J
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011522:Protons; D012366:RNA, Transfer, Val / Q000378:metabolism*; D013696:Temperature
D011522:Protons; D012366:RNA, Transfer, Val; D008274:Magnesium