Lead(II) cleavage analysis of RNase P RNA in vivo.
The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.
Lindell
Magnus
M
Department of Cell and Molecular Biology, Uppsala University, Box 596, S-75124 Uppsala, Sweden.
Brännvall
Mathias
M
Wagner
E Gerhart H
EG
Kirsebom
Leif A
LA
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism* / Q000502:physiology; D007854:Lead / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P / Q000378:metabolism* / Q000502:physiology; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D007854:Lead; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli