PMID:12756331
| Citation |
Zahler, NH, Christian, EL and Harris, ME (2003) Recognition of the 5' leader of pre-tRNA substrates by the active site of ribonuclease P. RNA 9:734-45 |
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| Abstract |
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. |
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| Keywords |
5' Untranslated Regions/metabolism; Adenosine/genetics; Base Sequence; Binding Sites; Catalysis; Conserved Sequence; Endoribonucleases/chemistry; Endoribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Models, Biological; Mutation; RNA Precursors/chemistry; RNA Precursors/metabolism; RNA, Archaeal/chemistry; RNA, Archaeal/metabolism; RNA, Bacterial/chemistry; RNA, Bacterial/metabolism; RNA, Catalytic/chemistry; RNA, Catalytic/metabolism; RNA, Transfer/chemistry; RNA, Transfer/metabolism; Ribonuclease P; Sequence Analysis, RNA; Substrate Specificity |
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