Kinetic Mechanism of Bacterial RNase P
This chapter analyzes the functional contributions of the three components required for bacterial RNase P catalysis: PRNA, P protein, and magnesium ions. This comprehensive overview of the bacterial RNase P reaction analyzes the kinetic data demonstrating a minimal kinetic mechanism with diffusion-controlled substrate association, rapid bond cleavage, and slow product release. The possibility of an additional step, a conformational change following substrate binding, in the minimal mechanism is also addressed. The kinetics of pre-tRNA 5′ cleavage catalyzed by the bacterial PRNA ribozyme, bacterial RNase P holoenzyme, and yeast RNase P holoenzyme are carefully compared.
KeywordsKinetic Mechanism Hepatitis Delta Virus Association Rate Constant Single Turnover Central Cleft
We would like to thank Nathan Zahler, Terry Watt, and James Hougland for their helpful discussions in the preparation of this manuscript. This project is supported National Institutes of Health (GM 55387 (CAF) and T32 GM08353 (KSK)).
- Cassano AG, Anderson VE, Harris ME (2004a) Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis. Biochemistry 43(32):10547–10559CrossRefPubMedGoogle Scholar
- Fersht A (1985) Enzyme structure and mechanism. W.H. Freeman, New YorkGoogle Scholar
- Hougland JL, Piccirilli JA, Forconi M, Lee J, Herschlag D (2006) How the group I intron works: a case study of RNA structure and function. In: Gesteland RF, Cech T, Atkins JF (eds) The RNA world. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- Hsieh J, Fierke CA (2009). Conformational change in the B. subtilis RNase P-pre-tRNA complex enhances substrate affinity and limits cleavage rate. RNA 15(8):1565–1577Google Scholar
- Hsieh J, Walker S, Fierke CA, Engelke DR (2009) Pre-tRNA cleavage by the yeast nuclear RNase P holoenzyme is rate-limited by slow product release. RNA 15(2):224–234Google Scholar
- Peck-Miller KA, Altman S (1991) Kinetics of the processing of the precursor to 4.5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli. J Mol Biol 221(1):1–5Google Scholar
- Rox C, Feltens R, Pfeiffer T, Hartmann RK (2002) Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme. J Mol Biol 315(4):551–560Google Scholar
- Serpersu EH, Shortle D, ASM (1986) Kinetic and magnetic resonance studies of effects of genetic substitution of a Ca2 + -liganding amino acid in staphylococcal nuclease. Biochemistry 25(1):68–77Google Scholar
- Sun L, Harris ME (2007) Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity. RNA 13(9):1505–1515Google Scholar
- Zahler NH, Koutmou KS, Kurz JC, Campbell FE, Harris ME, Fierke CA (2009) Protein-substrate contact leads to recognition of 5’ leaders by bacterial RNase P. (submitted)Google Scholar