Dissecting Functional Cooperation among Subunits in a Catalytic Ribonucleoprotein

核糖核酸酶P(RNase P)是一种将前体tRNAs(PtRNAs)加工成成熟形式所必需的酶。RNaseP全酶是一种核糖核蛋白(RNP)，在细菌、古生菌和真核生物中分别由一个必需的催化RNaseP RNA(RPR)和至少一个、四个和九个RNaseP蛋白(RPP)组成。这项研究的长期目标是了解古生代RNaseP中单个催化RNA和多个蛋白质辅助因子之间的动态功能相互作用，RNaseP是其真核亲缘的替代品，迄今已被证明在生化上是难处理的。我们将追求两个具体目标。首先，由于目前已知的古生代RPP是根据它们与真核生物近亲的同源性进行计算鉴定的，因此有必要通过实验揭示与天然RNaseP相关的所有亚基。因此，将从嗜热的kodakarasis和中温的maripaludis中分离出核糖核酸酶P，并对其亚基组成进行鉴定。具体地说，同源重组将被利用来用亲和标记的版本取代染色体上的RPP基因，从而提供一个句柄来纯化天然的RNaseP全酶。其次，将利用动力学和热力学研究不同的古生菌RPR是否有RPP，以剖析单个RPP对底物结合、切割速度和加工保真度的贡献。此外，RPP增强活性中心金属离子亲和力的能力将通过使用一种底物来描述，在该底物中，RNaseP裂解中心的金属配位氧被硫取代。这种原子取代使底物可与Cd~(2+)而不是Mg~(2+)切割，从而提供了一种策略，以磨练仅用于催化(Cd~(2+))而不是RPR折叠(Mg~(2+))的金属离子。这些结果将与报道的细菌RNase P的结果进行比较，在细菌RNase P中，单个RPP能够以几乎相同的效率对不同的ptRNAs和生理镁浓度进行RPR催化。这种比较有望为招募多个RPP是否为古生菌RNaseP全酶提供优势提供见解。更广泛的影响：全面了解细胞代谢需要充分了解所有对生存至关重要的酶，如RNaseP。通过提供对RNaseP催化中RNA-蛋白质相互作用的见解，该项目的结果将作为研究其他细胞RNPs中类似功能协调的模型。此外，对RNaseP的研究将有助于我们理解具有信息内容以外的功能的RNAs，并提供它们从RNA世界到现有的以蛋白质为主的细胞进化的快照。RNaseP是早期代谢的罕见残留物，被认为是由RNA酶主导的。项目实习生(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有关的基本机械问题，以及生物化学、酶学和分子生物学的现代技术。该项目的进展将适当地纳入俄亥俄州立大学和其他地方开设的核酸生物化学课程。作为NSF REU站点项目的联合主任，PI一直积极从代表性不足的群体中招收学生，并将寻求增加多样性，促进少数族裔参与这一项目。国际和平协会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推广活动提供了一个智力平台。

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.