Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release.
Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5' maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3' trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.
Hsieh
John
J
Department of Chemistry, University of Michigan, Ann Arbor, 48109-0606, USA.
Walker
Scott C
SC
Fierke
Carol A
CA
Engelke
David R
DR
D002384:Catalysis; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D020035:Holoenzymes; D012322:RNA Precursors; D029701:Saccharomyces cerevisiae Proteins; D043262:Ribonuclease P