Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P.
The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a approximately 400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5' leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5' of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2' deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.
Zahler
Nathan H
NH
Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4973, USA.
Christian
Eric L
EL
Harris
Michael E
ME
D020121:5' Untranslated Regions / Q000378:metabolism*; D000241:Adenosine / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity
D020121:5' Untranslated Regions; D029968:Escherichia coli Proteins; D012322:RNA Precursors; D019642:RNA, Archaeal; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D000241:Adenosine