Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109, USA.
J Mol Biol. 2010 Feb 12;396(1):195-208. doi: 10.1016/j.jmb.2009.11.039. Epub 2009 Nov 28.
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 pre-tRNA 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 pre-tRNA 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.
细菌核糖核酸酶 P（RNase P）催化前体 tRNA（pre-tRNA）5' 前导序列的切割。在此之前，该系统中所有已知的底物核苷酸特异性均来自于与 RNase P RNA 亚基的 RNA-RNA 相互作用。在这里，我们证明了枯草芽孢杆菌和大肠杆菌 RNase P 对 pre-tRNA 的结合亲和力通过切割位点 5' 侧的第四位前导核苷酸（N(-4)）与 RNase P 蛋白（P 蛋白）亚基之间的序列特异性相互作用而增强。枯草芽孢杆菌 RNase P 对 N(-4) 处为腺嘌呤的 pre-tRNA 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得到放大。对 N(-4) 处含有 pre-tRNA 的腺嘌呤类似物的测量表明，特异性源于与腺嘌呤的 N6 外环胺的氢键结合以及鸟嘌呤的 N2 胺的空间排斥。枯草芽孢杆菌 P 蛋白的突变表明 F20 和 Y34 对 N(-4) 处的选择性有贡献。Y34 的羟基通过与 N(-4) 核苷酸形成氢键来增强选择性。大肠杆菌 RNase P 的序列偏好性降低，表明 N(-4) 处对腺嘌呤和胞嘧啶有较弱的偏好，这与大肠杆菌 P 蛋白中 Leu 取代 Y34 一致。这是首次鉴定出 P 蛋白与 pre-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 pre-tRNA 中有超过预期比例的 pre-tRNA 在 N(-4) 处含有增强 RNase P 亲和力的核苷酸。这一观察结果表明，N(-4) 处的特异性有助于体内的底物识别。此外，生物信息学分析表明，蛋白质亚基与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，并可能导致物种特异性的底物识别。
