Probing structural differences between native and in vitro transcribed Escherichia coli valine transfer RNA: evidence for stable base modification-dependent conformers.
Structural differences between native (modified) and in vitro transcribed (unmodified) Escherichia coli tRNA(Val) were explored by comparing their temperature-absorbance profiles as a function of magnesium ion concentration and by probing their solution conformation with single- and double-strand-specific endonucleases. In vitro transcribed tRNA(Val) has a less ordered structure as monitored by thermal melting profiles; its Tm is appreciably lower than that of native tRNA(Val) at all Mg2+ concentrations. Structure probing experiments with nuclease S1 and ribonuclease V1 show that the unmodified tRNA(Val) transcript is more susceptible to nuclease attack at low Mg2+ concentrations, particularly in the D- and T-loops, indicative of at least a partial disruption of D-loop/T-loop interactions. These experiments also provide evidence for temperature-dependent alternative conformations of the anticodon loop of native tRNA(Val). Modified nucleosides are essential for the stability of these conformers; they cannot be detected in the unmodified in vitro transcript. The observations suggest that post-transcriptional modifications in tRNA allow the adoption of unique conformations and act to stabilize those that are biologically active.
Derrick
W B
WB
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Horowitz
J
J
D001483:Base Sequence; D004722:Endoribonucleases; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012366:RNA, Transfer, Val / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D014158:Transcription, Genetic
D012366:RNA, Transfer, Val; D004722:Endoribonucleases; C032825:ribonuclease V(1); D008274:Magnesium