Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination.
We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.
Brännvall
Mathias
M
Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-75124 Uppsala, Sweden.
Pettersson
B M Fredrik
BM
Kirsebom
Leif A
LA
D001483:Base Sequence; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D008345:Manganese; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium