Investigation of the catalytic mechanism of RNase P: the role of divalent metal ions and functional groups important for catalysis

The ribonucleoprotein enzyme ribonuclease (RNase) P is an endonuclease that generates the mature 5' -ends of tRNA. Bacterial RNase P is composed of a large RNA subunit (P RNA) and a small protein (P protein). Studies with P RNA from E. coli and B. subtilis have implied a specific role for two o...

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Main Author: Cuzic-Feltens, Simona-Daniela
Contributors: Hartmann, Roland K. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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Summary:The ribonucleoprotein enzyme ribonuclease (RNase) P is an endonuclease that generates the mature 5' -ends of tRNA. Bacterial RNase P is composed of a large RNA subunit (P RNA) and a small protein (P protein). Studies with P RNA from E. coli and B. subtilis have implied a specific role for two or more metal ions in substrate binding and cleavage chemistry. In this study it is demonstrated, for the first time, catalysis by E. coli P RNA with zinc as the sole divalent metal ion cofactor. Although proficient in catalysis, zinc destabilises E•S complex formation. In contrast, strontium inhibits catalysis, but promotes high substrate affinity. Stimulating and inhibitory effects of strontium could be rationalised by a model involving two strontium ions (or two classes), both improving substrate affinity in a cooperative manner, but one of the two inhibiting substrate conversion in a noncompetitive mode with respect to substrate. Further analyses suggest that the inhibition mode of strontium is noncompetitive with respect to zinc. The 2'-OH group at the scissile phosphodiester (nt –1 of ptRNA) contributes to positioning of a catalytic metal ion and may donate a H-bond to the 3'-O leaving group. NMR analyses have indicated that the ribose at nt –1 predominantly populates the C2'-endo conformation. Since the energy barrier for interconversion of C2'- and C3'-endo puckering is expected to be low, it is unknown which conformation is adopted during P RNA catalysis. To address this issue, we analysed cleavage of a ptRNA carrying an locked nucleic acid (LNA) substitution at nt –1, LNA being the only well known substituent that locks the ribose in a C3'-endo puckering. A 2'-methoxy substitution at this position was analysed in parallel, since it is chemically closely related to LNA. Other variants with 2'-fluoro or 2'-deoxy substitution at nt –1 were included in this study. An LNA substitution at nt –1 dramatically reduced (more that a 2'-deoxy) cleavage at the canonical site (-1/+1), while the effects on ground state binding were marginal. A 2'-methoxy substitution completely abolished cleavage at the canonical site. Also, both substituents suppressed cleavage at the site -1/-2. Instead, aberrant cleavage at the site +1/+2 was observed. Since the cleavage at the canonical site of the substrate with LNA at nt –1 had a higher magnesium requirement compared to cleavage at the +1/+2 site, it is likely that the methylene group of LNA inhibit cleavage at the canonical site by sterical interference with a catalytic magnesium ion. The fact that LNA at nt –1 still permitted residual cleavage at the canonical site indicates that the transition state can be reached in the presence of a locked C3'-endo conformation at nt –1. We further tested LNA at nt +1. Here, LNA had only a marginal effect on cleavage chemistry, but significantly reduced ptRNA binding affinity. The binding defect could be overcome at high metal ion concentrations. Again, comparison with a 2'-methoxy and 2'-deoxy modification indicated sterical hindrance by the methylen or methyl group. Hill analysis of metal ion dependence of ptRNA binding revealed a higher metal ion cooperativity for the LNA variant compared to the unmodified one, indicating that the methylene group sterically interferes, directly or indirectly, with metal ion binding to at least one site crucial for E•S complex formation. Functional groups within the P RNA and/or ptRNA are essential for substrate binding and cleavage and they can be mapped by modification interference studies: nucleotide analogue interference mapping (NAIM) and suppression (NAIS). For this purpose, an RNA chimera consisting of E. coli P RNA and the tRNA 5'-half was constructed. A functional substrate was reconstituted by annealing the tRNA 5'-half with its 3'-half resulting in a cis-cleaving RNA complex. A partially modified RNA pool (RNA chimera) was then synthesised by in vitro transcription. After separation of functional (cis-cleaving) and non-functional molecules, positions within the 3' -half of the P RNA where modifications interfered with processing (cis-cleavage reaction) could be identified. These positions are overlapping to some extent with those found in a previous study, where interference with E. coli P RNA - tRNA binding was analysed. These results are to be expected because functional groups in P RNA important for substrate binding are likewise important for substrate turnover, since binding is a prerequisite for cleavage. A strong interference effect at G 350 was detected only with the cis – cleavage assay. G 350 may represent a position essential for the catalytic step, as evidence was provided that it contributes to the binding of catalytically important magnesium near the active site of P RNA.
Physical Description:150 Pages