Role of ribozymes and enzymes in tRNA processing.
Ribonuclease P (RNase P) is the enzyme responsible for cleaving the 5′-leader sequence from precursor tRNA molecules. In bacteria, the catalytic subunit of RNase P is an RNA molecule or ribozyme ~400 nucleotides long; and in all other organisms studied, the enzyme also contains a catalytically-required RNA subunit. In contrast, we find that RNase P from plant chloroplasts appears to be a single protein. Chloroplast RNase P also uses different catalytic chemistry than the E. coli RNase P ribozyme. tRNA cleavage by the E. coli enzyme absolutely requires Mg2+ bound to the pro-RP oxygen of the scissile phosphodiester; the metal probably generates the hydroxide ion which hydrolyzes the bond. In contrast, chloroplast RNase P does not utilize a metal ion at that position. We are currently pursuing protein chemistry and genetic approaches to identify the chloroplast enzyme, and we are investigating whether other groups surrounding the scissile bond help bind metal ions during the RNase P reaction.
Our lab is also collaborating with Dr. Mark Richter's group to investigate how structural interactions among the subunits of chloroplast ATP synthase generate its novel allosteric and regulatory properties. To this end, we have cloned, genetically altered, over-expressed, and refolded the five polypeptides of the catalytic CF1 component and partially reconstituted them in vitro. A heterologous in vivo reconstitution system allowed us to define amino acids at a subunit interface which are required for cooperative synthesis of ATP. We are now developing systems that will allow us to determine how the g subunit regulates and confers cooperativity on the catalytic a and b subunits.
- Samra HS, Gao F, He F, Hoang E, Chen Z, Gegenheimer PA, Berrie CL, Richter ML. Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit. J Biol Chem. 2006 Oct 13;281(41):31041-9.
- Gegenheimer P. Enzyme nomenclature: functional or structural? RNA. 2000 Dec;6(12):1695-7.
- Thomas BC, Chamberlain J, Engelke DR, Gegenheimer P. Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P. RNA. 2000 Apr;6(4):554-62.
- Thomas BC, Li X, Gegenheimer P. Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism. RNA. 2000 Apr;6(4):545-53.
- Sokolov M, Lu L, Tucker W, Gao F, Gegenheimer PA, Richter ML. The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity. J Biol Chem. 1999 May 14;274(20):13824-9.
- Chen Y, Li X, Gegenheimer P. Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond. Biochemistry. 1997 Mar 4;36(9):2425-38.
- Gegenheimer, P. (1996) Structure, mechanism, and evolution of chloroplast transfer RNA processing systems. Mol. Biol. Rep. 2/3, 147–150.
- Thomas, B.C., Gao, L., Stomp, D., Li, X., and Gegenheimer, P. (1995) Spinach chloroplast RNase P: a putative protein enzyme. Nucleic Acids Symp. Series 33, 95–98.
- Chen, Z., Spies, A., Hein, R., Zhou, X., Thomas, B., Richter, M. L., and Gegenheimer, P. (1995) A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes. J. Biol. Chem. 270, 1–9.
- Oommen, A., Li, X., and Gegenheimer, P. (1992) Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases. Mol. Cell. Biol. 12, 865–875.
- Wang, M.-J., Davis, N. W., and Gegenheimer, P. (1988) Novel mechanisms for maturation of chloroplast transfer RNA precursors. EMBO J. 7, 1567–1574.
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