Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P.
Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
Darr
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Department of Biology, Indiana University, Bloomington 47405.
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Smith
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D001483:Base Sequence; D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D043262:Ribonuclease P; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli