Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.
Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.
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DOI：
10.1093/nar/gkq732
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发表时间：
2011-02
影响因子：
14.9
通讯作者：
Kirsebom LA
中科院分区：
文献类型：
--
作者：
Sinapah S;Wu S;Chen Y;Pettersson BM;Gopalan V;Kirsebom LA
RNase P是一种催化核糖核蛋白，主要参与tRNA的生物合成。核糖核酸酶P（RNase P）由催化性RNase P RNA（RPR）和至少四种蛋白质辅因子（RPP）组成，它们以两种二元复合物（POP 5·RPP 30和RPP 21·RPP 29）起作用。利用在体外组装功能性激烈火球菌（Pfu）RNase P的能力，我们研究了RPPs在影响RPR底物识别中的作用。我们首先证明，Pfu RPR，像它的细菌和真核生物的同行，切割模型发夹环底物，虽然在率90- 200倍低时，与裂解细菌RPR相比，突出了功能相当的催化核心细菌和古细菌RPR。通过研究Pfu RPR（±RPPs）与各种缺失共识识别元件的模型底物表现出的裂解位点选择，我们确定了POP 5·RPP 30或RPP 21·RPP 29（直接或间接通过RPR）促进识别的底物特征。我们的研究结果还表明，Pfu RPR + RPP 21·RPP 29显示出与细菌RPR单独反应而不是Pfu RPR的底物识别特性相一致的底物识别特性，并且这种行为可归因于细菌和古细菌RPR的底物特异性结构域的结构差异。此外，我们的数据揭示了一个层次结构的识别元件，决定裂解位点的选择古细菌RNase P。
RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.
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