Talk abstracts

Talk on Saturday 11:05-11:20am submitted by Wen Yi Chen

Use of archaeal RNase P as a model system to dissect the roles of protein cofactors in aiding RNA catalysis

Wen-Yi Chen (Molecular, Cellular, and Developmental Biology Program and Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA), Venkat Gopalan (Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA)

Abstract:
Ribonuclease P (RNase P) is a Mg²⁺-dependent endoribonuclease which aids tRNA maturation by removing the 5' leader of precursor tRNA(1). In bacteria, the enzyme contains a catalytic RNase P RNA (RPR) and one RNase P protein (RPP). In archaea and eukarya, RNase P contains an indispensable RPR with at least four and nine RPPs, respectively. However, only few euryarchaeal and eukaryal RPRs are catalytically active in vitro(2, 3) despite remarkable secondary structure similarity with bacterial RPRs(4-7). This decreased archaeal/eukaryal RPR function may reflect an evolutionary path towards a more complex RNP in which RPPs contribute to structural stabilization or provide functional groups for catalysis. By using recombinant archaeal RPR and four RPPs, we have successfully reconstituted the holoenzyme (i.e., RPR-4RPPs)(8) and have used single-turnover kinetic measurements to focus on the phosphodiester bond-breaking step. Interestingly, when archaeal RPR was reconstituted with binary complexes of RPPs, only POP5-RPP30, but not RPP21-RPP29, was able to enhance k_obs for the archaeal RPR by ~20-fold. However, addition of RPP21-RPP29 to a ternary complex (RPR-POP5-RPP30) results in an additional 5-fold increase in k_obs. We also found that the four archeal RPPs can effectively rescue mutant RPRs, in which active site metal ion binding is weaken(9). We are now attempting to determine which binary complex is instrumental for the rescue presumably through enhancing the RPR’s ability to bind Mg²⁺ ions. Lastly, since the RPP21-RPP29 pair was able to lower the RPR’s requirement for monovalent and divalent ion concentrations, it is likely involved in capturing an RPR tertiary fold necessary for function. Taken together, our studies are beginning to delineate the distinctive roles of RPPs in archaeal RNase P catalysis and will serve as a valuable paradigm for understanding functional interplay among subunits in catalytic RNP complexes

References:
1. Altman S. 2007. Mol Biosyst 3: 604-7
2. Pannucci JA, Haas ES, Hall TA, Harris JK, Brown JW. 1999. Proc Natl Acad Sci U S A 96: 7803-8
3. Kikovska E, Svard SG, Kirsebom LA. 2007. Proc Natl Acad Sci U S A 104: 2062-7
4. Chen JL, Pace NR. 1997. RNA 3: 557-60
5. Haas ES, Armbruster DW, Vucson BM, Daniels CJ, Brown JW. 1996. Nucleic Acids Res 24: 1252-9
6. Frank DN, Adamidi C, Ehringer MA, Pitulle C, Pace NR. 2000. RNA 6: 1895-904
7. Brown JW. 1999. Nucleic Acids Res 27: 314
8. Tsai HY, Pulukkunat DK, Woznick WK, Gopalan V. 2006. Proc Natl Acad Sci U S A 103: 16147-52
9. Kaye NM, Zahler NH, Christian EL, Harris ME. 2002. J Mol Biol 324: 429-42

Keywords: Ribonuclease P, Archaeal RPP, Single-turnover