Kikovska Ema, Brännvall Mathias, Kirsebom Leif A
Department of Cell and Molecular Biology, Uppsala University, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.
J Mol Biol. 2006 Jun 9;359(3):572-84. doi: 10.1016/j.jmb.2006.03.040. Epub 2006 Apr 3.
Most tRNAs carry a G at their 5' termini, i.e. at position +1. This position corresponds to the position immediately downstream of the site of cleavage in tRNA precursors. Here we studied RNase P RNA-mediated cleavage of substrates carrying substitutions/modifications at position +1 in the absence of the RNase P protein, C5, to investigate the role of G at the RNase P cleavage site. We present data suggesting that the exocyclic amine (2NH2) of G+1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage. This is in contrast to O6, N7 and 2'OH that are suggested to affect ground state binding and rate of cleavage to significantly lesser extent. We also provide evidence that the effects caused by the absence of 2NH2 at position +1 influenced the charge distribution and conceivably Mg2+ binding at the RNase P cleavage site. These findings are consistent with models where the 2NH2 at the cleavage site (when present) interacts with RNase P RNA and/or influences the positioning of Mg2+ in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at +1 is not essential for cleavage but its presence suppresses miscleavage and dramatically increases the rate of cleavage. Together our findings provide reasons why most tRNAs carry a guanosine at their 5' end.
大多数tRNA在其5'末端（即+1位置）携带一个鸟嘌呤（G）。该位置对应于tRNA前体中切割位点下游紧邻的位置。在此，我们研究了在不存在核糖核酸酶P蛋白C5的情况下，核糖核酸酶P RNA对在+1位置携带取代/修饰的底物的切割作用，以探究G在核糖核酸酶P切割位点的作用。我们提供的数据表明，G+1的环外氨基（2NH2）通过影响切割速率，有助于切割位点识别、基态结合和催化作用。这与O6、N7和2'OH形成对比，它们被认为对基态结合和切割速率的影响程度要小得多。我们还提供证据表明，+1位置缺少2NH2所产生的影响会改变电荷分布，并可能影响核糖核酸酶P切割位点处Mg2+的结合。这些发现与以下模型一致：切割位点处的2NH2（如果存在）与核糖核酸酶P RNA相互作用和/或影响切割位点附近Mg2+的定位。此外，我们的数据表明，+1位置碱基的存在对于切割并非必不可少，但它的存在会抑制错误切割并显著提高切割速率。我们的研究结果共同揭示了为什么大多数tRNA在其5'末端携带鸟苷。
