A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3
A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3
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DOI：
10.1016/j.bbrc.2006.02.192
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发表时间：
2006-05-12
影响因子：
3.1
通讯作者：
Kimura, M
中科院分区：
文献类型：
--
作者：
Fukuhara, H;Kifusa, M;Kimura, M
核糖核酸酶P（RNase P）是一种核糖核蛋白复合物，参与前体tRNA的5'前导序列的加工。我们以前发现，重组颗粒（RP）由RNase P RNA和四种蛋白质组成，（Ph 1481 p、Ph 1601 p、Ph 1771 p和Ph 1877 p）在超嗜热古菌Pyrococcus horikoshii OT 3中表现出RNase P活性，但最适温度较低（大约在55 ℃），而来自P. horikoshii的真实RNase P为70 ℃ [Kouzuma等人，生物化学、生物物理Res. Commun. 306（2003）666-673]。在目前的研究中，我们发现添加第五种蛋白Ph 1496 p，一种假定的核糖体蛋白L7 Ae，使用热位移测定和化学探针定位的表征将Ph 1496 p结合位点定位在两个茎-环结构上，包括核磷脂A116-G201和G229-G229。C276在堀越伪霉RNase P RNA中的表达。此外，Ph 1496 p的晶体结构，确定在2.0埃分辨率的分子置换法使用核糖体蛋白L7 Ae从Haloarcula marismortui作为搜索模型。ph 1496 p包括五个。螺旋和一个四股β-连接体β-折叠被夹在一侧的三个螺旋（α 1、α 4和α 5）和另一侧的两个螺旋（α 2和α 3）之间。已知古细菌核糖体蛋白L7 Ae是三重功能蛋白质，作为核糖体和核糖核蛋白复合物、盒C/D和盒H/ACA中的蛋白质组分。虽然我们目前还没有直接证据表明Ph 1496 p是堀越氏毕赤酵母RNase P中的一个真实的蛋白组分，但本研究结果可能将RNase P蛋白作为第四种功能分配给L7 Ae。(c)2006年爱思唯尔公司All rights reserved.
Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et A., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using get shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nuclecitides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 angstrom resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five of.-helices and a four stranded beta-shect. The beta-sheet is sandwiched by three helices (alpha 1, alpha 4, and alpha 5) at one side and two helices (alpha 2 and alpha 3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function. (c) 2006 Elsevier Inc. All rights reserved.