Dissecting Functional Cooperation among Subunits in a Catalytic Ribonucleoprotein

核糖核酸酶P（RNase P）是将前体tRNA（ptRNA）加工成其成熟形式所必需的酶。RNase P全酶是一种核糖核蛋白（RNP），在细菌、古细菌和真核生物中分别包含一种必需的催化RNase P RNA（RPR）和至少一种、四种和九种RNase P蛋白（RPP）。这项研究的一个长期目标是了解古细菌RNase P中单个催化RNA和多个蛋白质辅因子之间的动态功能相互作用，RNase P是其真核相对物的替代物，迄今为止已被证明是生物化学上难以解决的。将追求两个具体目标。首先，由于目前已知的古细菌RPPs是基于其与真核亲缘的同源性通过计算鉴定的，因此有必要通过实验揭示与天然RNase P相关的所有亚基。因此，RNase P从嗜热Thermococcus kodakaraensis和嗜温Methanococcus maripaludis将被分离和其特征在于相对于它们的亚基组成。具体地，将利用同源重组来用亲和标记的版本替换染色体RPP基因，从而提供纯化天然RNase P全酶的手柄。第二，动力学和热力学研究不同的古细菌RPR与和没有RPPs将被用来解剖的贡献，个别RPPs基板结合，裂解率，和保真度的处理。此外，RPPs增强活性位点金属离子亲和力的能力将通过使用其中RNA酶P切割位点处的金属配位氧被硫取代的底物来描绘。这样的原子取代使得底物可被Cd 2+而不是Mg 2+裂解，从而提供了一种策略来磨练仅用于催化（Cd 2+）而不是RPR折叠（Mg 2+）所需的金属离子。这些结果将与细菌RNase P的报道进行比较，其中单个RPP使得RPR催化对于不同的ptRNA和生理Mg 2+浓度具有几乎相等的效率。这样的比较，预计提供的见解是否招聘多个RPPs赋予优势的古细菌RNase P全酶。更广泛的影响：对细胞代谢的全面认识需要对所有对生存能力至关重要的酶（如RNase P）进行全面了解。通过深入了解RNase P催化中的RNA-蛋白质相互作用，该项目的结果将作为其他细胞RNP中类似功能协调的研究模型。此外，对RNase P（一种被怀疑由RNA酶主导的早期代谢的罕见残留物）的研究将有助于我们理解RNA的功能，而不是信息内容，并提供它们从RNA世界进化到现有蛋白质主导细胞的快照。项目学员（本科生，研究生和博士后学者）将接触到与独特的生物催化剂和生物化学，酶学和分子生物学现代技术相关的基本机械问题。该项目的进展将适当地纳入俄亥俄州州立大学和其他地方提供的核酸生物化学课程。PI作为NSF REU网站计划的联合主任，一直积极招募代表性不足的群体的学生，并将寻求增加多样性和促进少数民族参与该项目。PI的教学和研究兴趣也为以短期课程和讲习班的形式开展国际外联活动提供了知识平台。

Ribonuclease P (RNase P) is an enzyme essential for processing precursor tRNAs (ptRNAs) to their mature forms. The RNase P holoenzyme is a ribonucleoprotein (RNP) comprising one essential catalytic RNase P RNA (RPR) and at least one, four, and nine RNase P proteins (RPPs) in bacteria, archaea and eukarya, respectively. A long-term goal of this research is to understand the dynamic functional interplay among the single catalytic RNA and multiple protein cofactors in archaeal RNase P, a surrogate for its eukaryal relative that has proven biochemically intractable so far. Two specific aims will be pursued. First, since currently known archaeal RPPs were computationally identified based on their homology to eukaryal relatives, experimentally uncovering all subunits associated with native RNase P is necessary. Therefore, RNase P from the thermophilic Thermococcus kodakaraensis and mesophilic Methanococcus maripaludis will be isolated and characterized with respect to their subunit make-up. Specifically, homologous recombination will be exploited to replace a chromosomal RPP gene with an affinity-tagged version thus providing a handle to purify the native RNase P holoenzyme. Second, kinetic and thermodynamic studies on different archaeal RPRs with and without RPPs will be used to dissect the contributions of individual RPPs to substrate binding, cleavage rate, and fidelity of processing. Furthermore, the ability of RPPs to enhance affinity for active-site metal ions will be delineated by using a substrate in which the metal-coordinating oxygen at the RNase P cleavage site is substituted with sulfur. Such an atomic substitution renders the substrate cleavable with Cd2+ and not Mg2+, thus providing a strategy to hone in on metal ions required solely for catalysis (Cd2+) and not RPR folding (Mg2+). These results will be compared with those reported for bacterial RNase P, where a single RPP enables RPR catalysis with near-equal efficiency for different ptRNAs and at physiological Mg2+ concentrations. Such a comparison is expected to furnish insights into whether recruitment of multiple RPPs conferred advantages to the archaeal RNase P holoenzyme. Broader impacts: An overall appreciation of cellular metabolism requires a full understanding of all enzymes that are essential for viability such as RNase P. By furnishing insights into the RNA-protein interplay in RNase P catalysis, results from this project will serve as a model for investigations of similar functional coordination in other cellular RNPs. Moreover, studies on RNase P, a rare remnant of the early metabolism suspected to be dominated by RNA enzymes, will contribute to our understanding of RNAs with functions other than those of information content and offer snapshots of their evolution from the RNA world to the existing protein-dominated cell. Project trainees (undergraduates, graduates, and postdoctoral scholars) will be exposed to fundamental mechanistic questions related to a unique biocatalyst and to modern techniques in biochemistry, enzymology, and molecular biology. Advances from this project will be appropriately integrated into coursework on nucleic acid biochemistry to be offered at The Ohio State University and elsewhere. The PI, as co-director of an NSF REU site program, has been active in recruitment of students from under-represented groups and will seek to increase diversity and promote minority participation in this project. The PI's teaching and research interests have also provided an intellectual platform for developing international outreach activities in the form of short courses and workshops.