Poly(A) polymerase I of Escherichia coli:: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation
Poly(A) polymerase I of Escherichia coli:: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation
复制标题
DOI：
10.1046/j.1365-2958.1999.01394.x
复制
发表时间：
1999-05-01
影响因子：
3.6
通讯作者：
Carpousis, AJ
中科院分区：
文献类型：
--
作者：
Raynal, LC;Carpousis, AJ
大肠杆菌多聚(A)聚合酶I(PAP I)是核苷酸转移酶(NTR)超家族的成员，包括真核细胞的PAPs和所有已知的tRNACCA加入酶。PAP I的五个高度保守的天冬氨酸被改变为丙氨酸或脯氨酸，证明了它们对聚合酶活性的重要性。在所有NTR中绝对保守的甘氨酸也被改变，产生了一种新的突变蛋白，其中ATP在不依赖于引物的反应中被浪费地水解，这是第一次表征真细菌PAP的催化部位的工作，尽管某些序列是保守的，但我们预测真核细胞和真细菌活性部位的整体结构可能不同。用截短形式的PAP I通过Northwest和Far-Western blotting绘制了RNase E与RNA的结合部位图，发现PAP I与CSDA、RhlE和SrmB之间还有蛋白质-蛋白质相互作用，这表明PAP I与这些大肠杆菌DEAD box RNA解旋酶之间存在意想不到的联系，这些结果表明PAP I的功能组织类似于真核PAP，具有N端催化结构域、C端RNA结合域和与其他蛋白质因子相互作用的部位。
Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction, This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by Northwestern and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases, These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.