Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P
Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P
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DOI：
10.1016/j.jmb.2009.11.039
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发表时间：
2010-02-12
影响因子：
5.6
通讯作者：
Fierke, Carol A.
中科院分区：
文献类型：
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作者：
Koutmou, Kristin S.;Zahler, Nathan H.;Fierke, Carol A.
细菌核糖核酸酶 P (RNase P) 催化前体 tRNA (pre-tRNA) 的 5' 前导序列裂解。此前，该系统中所有已知的底物核苷酸特异性均源自 RNA-RNA 与 RNase P RNA 亚基的相互作用。在这里，我们证明 pretRNA 对枯草芽孢杆菌和大肠杆菌 RNase P 的结合亲和力通过切割位点 5' 侧的第四个 pre-tRNA 核苷酸 (N(-4)) 和 RNase P 蛋白 (P 蛋白) 亚基之间的序列特异性接触而增强。 B. subtilis RNase P 对 N(-4) 处腺苷的 pretRNA 具有更高的亲和力，并且这种结合偏好在生理二价离子浓度下被放大。对 N(-4) 处含有前 tRNA 的腺苷类似物的测量表明，特异性是由与腺苷 N6 环外胺的氢键结合和鸟苷 N2 胺的空间排斥的组合产生的。枯草芽孢杆菌 P 蛋白的诱变表明 F20 和 Y34 有助于 N(-4) 处的选择性。 Y34 的羟基可能通过与 N(-4) 核苷酸形成氢键来增强选择性。大肠杆菌 RNase P 的序列偏好减弱，显示出对 N (-4) 处腺苷和胞嘧啶的弱偏好，这与大肠杆菌 P 蛋白中用 Leu 替代 Y34 一致。这是首次鉴定出 P 蛋白和前 tRNA 之间的序列特异性接触，有助于 RNase P 的分子识别。此外，序列分析表明，来自大肠杆菌和枯草芽孢杆菌的前 tRNA 中含有高于预期的 N(-4) 核苷酸，可增强 RNase P 亲和力。这一观察结果表明，N(-4) 的特异性有助于体内底物识别。此外，生物信息学分析表明，蛋白质亚基和前 tRNA 前导序列之间的序列特异性接触可能在细菌 RNase P 中很常见，并且可能导致物种特异性底物识别。 (C) 2009 Elsevier Ltd. 保留所有权利。
Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pretRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pretRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N (-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition. (C) 2009 Elsevier Ltd. All rights reserved.