David M, Vekstein R, Kaufmann G
Proc Natl Acad Sci U S A. 1979 Nov;76(11):5430-4. doi: 10.1073/pnas.76.11.5430.
Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with [gamma-32P]ATP, 32P was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in the product polyribonucleotides was found in 5'-terminal phosphate groups, suggesting that they originated by a phosphorylation reaction catalyzed by the endogenous polynucleotide kinase (EC 2.7.1.78). Indeed, these products were not seen in an acellular system from uninfected cells, and their amount and complexity increased with the progress of infection. Analysis of the 32P-labeled polyribonucleotide products by gel electrophoresis, either before or after digestion with alkaline phosphatase (EC 3.1.3.1), revealed that a small fraction of the 32P resided in phosphodiester bonds of several tRNA-sized chains. This specific 32P transfer from [gamma-32P]ATP into phosphodiester bonds was apparently catalyzed by successive polynucleotide kinase and RNA ligase reactions. The possible relationship of the 32P transfer to RNA ligase was investigated next by using a system from cells infected with T4 am M69 (an amber mutant deficient in RNA ligase). Transfer of 32P from [gamma-32P]ATP into phosphodiester bonds was not detected in the am M69 system. However, addition of purified RNA ligase to the am M69 system restored the specific 32P transfer. A system from cells infected with T4 psu-b delta 33 (a deletion mutant lacking the entire tRNA region) sustained the specific 32P transfer into tRNA-sized products, indicating that they were not derived from transcripts of T4 tRNA genes. These data may reflect a role of RNA ligase in posttranscriptional conversion of presumably host polyribonucleotides into novel tRNA species during T4 infection.
为探寻T4 RNA连接酶[多聚核糖核苷酸合成酶（ATP）；多（核糖核苷酸）：多（核糖核苷酸）连接酶（形成AMP），EC 6.5.1.3]活性的生理作用，我们构建了一个受T4噬菌体感染的质壁分离大肠杆菌细胞的无细胞体系。将该体系与[γ-32P]ATP一起温育后，32P转移到大量多聚核糖核苷酸中，大多长达300 - 400个残基。在产物多聚核糖核苷酸中，大部分32P存在于5'-末端磷酸基团中，这表明它们起源于内源性多核苷酸激酶（EC 2.7.1.78）催化的磷酸化反应。实际上，在未感染细胞的无细胞体系中未观察到这些产物，并且随着感染进程它们的数量和复杂性增加。在用碱性磷酸酶（EC 3.1.3.1）消化之前或之后，通过凝胶电泳分析32P标记的多聚核糖核苷酸产物，发现一小部分32P存在于几条tRNA大小链的磷酸二酯键中。这种从[γ-32P]ATP到磷酸二酯键的特定32P转移显然是由连续的多核苷酸激酶和RNA连接酶反应催化的。接下来，通过使用来自感染T4 am M69（一种RNA连接酶缺陷的琥珀突变体）的细胞体系，研究了32P转移与RNA连接酶的可能关系。在am M69体系中未检测到32P从[γ-32P]ATP转移到磷酸二酯键中。然而，向am M69体系中添加纯化的RNA连接酶可恢复特定的32P转移。来自感染T4 psu-b delta 33（一个缺失整个tRNA区域的缺失突变体）的细胞体系维持了特定的32P转移到tRNA大小的产物中，表明它们并非源自T4 tRNA基因的转录本。这些数据可能反映了RNA连接酶在T4感染期间将推测的宿主多聚核糖核苷酸进行转录后转化为新的tRNA种类中的作用。
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