The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning.
The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning.
复制标题
DOI：
10.1093/nar/gks744
复制
发表时间：
2012-11-01
影响因子：
14.9
通讯作者：
Mondragón A
中科院分区：
文献类型：
--
作者：
Reiter NJ;Osterman AK;Mondragón A
RNaseP是一种基于RNA的酶，主要负责5‘端的前tRNA加工。细菌RNase P全酶与tRNAP形成的复合体的结构揭示了底物识别的结构基础，确定了活性部位，并显示了蛋白质组分如何增加功能。活性中心至少包括两个金属离子，一个通用尿苷(U52)和P RNA主干部分，但尚不清楚邻近的、细菌保守的蛋白质环(残基52-57)是否参与催化。在这里，突变与单周转反应动力学相结合表明，该环上的点突变对催化效率没有或略有影响。同样，代表细菌RNaseP蛋白最保守区域的‘RNR’区域的氨基酸变化对催化效率的影响可以忽略不计。然而，U52和两个细菌保守的蛋白残基(F17和R89)是有效的热致病杆菌RNase P活性所必需的。U52核苷酸在活性部位与金属离子结合，而F17和R89位于裂解部位>；20ä，可能与前tRNA5‘-先导的N−4和N−5核苷酸接触。这表明过渡态的形成和通过与引导者的相互作用而定位的衬底之间存在协同耦合。
RNase P is an RNA-based enzyme primarily responsible for 5′-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52–57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the ‘RNR’ region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N−4 and N−5 nucleotides of the pre-tRNA 5′-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.
登录
查看更多内容
影响因子：
2.9
作者：
Crary, SM;Niranjanakumari, S;Fierke, CA
通讯作者：
Fierke, CA
影响因子：
5.6
作者：
Kaye, NM;Zahler, NH;Harris, ME
通讯作者：
Harris, ME
影响因子：
15
作者：
Cassano, AG;Anderson, VE;Harris, ME
通讯作者：
Harris, ME
影响因子：
11.4
作者：
Buck, AH;Dalby, AB;Pace, NR
通讯作者：
Pace, NR
影响因子：
11.4
作者：
Christian, EL;Kaye, NM;Harris, ME
通讯作者：
Harris, ME