Pascual A, Vioque A
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Avenida Americo Vespucio s/n, 41092 Seville, Spain.
Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6672-7. doi: 10.1073/pnas.96.12.6672.
We have studied the effect of the 3' terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium Synechocystis sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3' terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in Escherichia coli RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330-332 in the J15/16 loop of Synechocystis RNase P RNA from GGU to CCA has a modest effect on kcat for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that Synechocystis RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3' terminal CCA is not involved.
我们在一个完全同源的系统中，研究了蓝藻集胞藻PCC 6803的tRNA前体中3'末端CCA序列对RNase P RNA或全酶催化作用的影响。我们发现，3'末端CCA的缺失对活性并无损害，这与其他细菌系统中的情况形成鲜明对比。我们还发现，在其他蓝藻中也是如此。这种情况与蓝藻中J15/16环的异常结构相关，J15/16环在大肠杆菌RNase P的CCA相互作用中是一个重要的环，同时也与蓝藻在基因组中不编码CCA序列而是在转录后添加它这一事实相关。将集胞藻RNase P RNA的J15/16环中330 - 332位核苷酸从GGU修饰为CCA，对含CCA底物的kcat有适度影响，对切割位点的选择没有影响。我们开发了一种直接的物理方法来检测RNase P RNA与其固定在滤膜上的底物之间的相互作用，并且我们确定集胞藻RNase P RNA与缺乏CCA的底物结合的亲和力比与含CCA的底物结合的亲和力更好。我们的结果表明，蓝藻RNase P中底物结合的模式不同于其他真细菌，且不涉及3'末端CCA。
