Identification of the coding region for a second poly(A) polymerase in Escherichia coli
Identification of the coding region for a second poly(A) polymerase in Escherichia coli
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DOI：
10.1073/pnas.93.21.11580
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Sarkar, N
中科院分区：
文献类型：
--
作者：
Cao, GJ;Pogliano, J;Sarkar, N
我们之前已经确定pcnB位点是大肠杆菌聚(A)聚合酶（PAP I）的主要基因。在这篇报告中，我们描述了通过实验策略破坏和鉴定第二聚(a)聚合酶（PAP II）的候选基因，该实验策略基于大肠杆菌的生存能力取决于PAP I或PAP II的存在的假设。由此确定的编码区为位于大肠杆菌染色体上约87 min的开放阅读框f310。以下证据支持f310是PAP II的基因：(i) f310编码的推断肽的分子量为36,300，类似于PAP II凝胶过滤估计的分子量为35,000；（ii）推导出的f310产物是一个相对疏水的多肽，pi为9.4，与部分纯化的PAP ii的性质一致；（iii） f310的过表达导致包涵体的形成，其溶解和恢复产生poly(A)聚合酶活性，对应于35kda的蛋白质，如酶印迹所示；（iv）在编码区n端表达f310与六组氨酸的融合构建体，可以纯化聚(a)聚合酶片段，其主要成分是36 kda蛋白。大肠杆菌PAP II与PAP I、病毒和真核生物的聚(A)聚合酶都没有明显的序列同源性，这表明细菌的聚(A)聚合酶是独立进化的。PAP II序列的一个有趣的特征是存在两组配对的半胱氨酸和组氨酸残基，它们类似于在其他一些蛋白质中看到的RNA结合基序。
We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pi of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.