Hsu T, Karam J D
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
J Biol Chem. 1990 Mar 25;265(9):5303-16.
A phage T4 genetic cluster that encodes DNA polymerase, several other DNA replication proteins, transcriptional factors, and the translation repressor RegA has been shown to be controlled by overlapping modes of transcription which initiate at several promoters. The promoters were mapped by using a combination of assays including Northern blotting, S1-mapping, RNA sequencing, and analysis of products of radioactive labeling of 5' ends on T4-induced RNA in vitro via the reaction catalyzed by eukaryotic guanylyl transferase (RNA capping assay). The most proximal in the cluster are two promoters that do not require any phage-induced factors for activation, i.e. they are T4 early promoters. Initiation at these promoters yields several RNA species having overlapping 5'-terminal sequences, the largest of which is estimated to be about 15,000 nucleotides long and to include all the cistrons of the cluster. A third early promoter maps inside the protein encoding segment of one of the cistrons (T4 gene 47), while at least five additional promoters map in intercistronic regions and are T4 middle promoters, i.e. they require the T4-induced DNA-binding transcription factor MotA. Transcriptional readthrough at a termination site within the T4 gene 45-44 intercistronic region is required for synthesis of gp44 and gp62, two essential T4 DNA-polymerase (gp43) accessory proteins. In contrast, transcription of T4 gene 43 is serviced by readthrough across a termination site in the regA-43 intercistronic region as well as by a MotA-dependent promoter that maps downstream of the termination site, and the region contains a site for processing by a T4-induced enzyme that also cleaves elsewhere in the polycistronic mRNA from the cluster (i.e. in the Shine-Dalgarno sequence of the gene 45.2 mRNA). The termination events in the gene 45-44 and regA-43 intercistronic regions both occur downstream of RNA stem-loop structures containing the sequence 5'CUUCGG3' in the loop segments. Transcription termination in the 78-base-pair regA-43 intercistronic region occurs about 60 nucleotides away from the gp43 initiator AUG, transcription initiation occurs at 38-40 nucleotides upstream from the AUG, and T4-dependent RNA processing occurs at several sites (including a GGAG sequence) between the transcription termination and initiation sites. Thus, all gp43-encoding mRNAs contain the translational operator (residues -40 to -1 relative to the AUG) for autogenous repression by this DNA polymerase (Andrake et al., 1988).(ABSTRACT TRUNCATED AT 400 WORDS)
一个编码DNA聚合酶、其他几种DNA复制蛋白、转录因子以及翻译阻遏物RegA的噬菌体T4基因簇，已被证明受多种转录起始模式的调控，这些转录起始于多个启动子。通过Northern印迹、S1作图、RNA测序以及真核鸟苷酸转移酶催化反应（RNA加帽分析）对T4诱导的体外RNA 5'端放射性标记产物进行分析等多种检测方法相结合，绘制了这些启动子的图谱。该基因簇中最靠近起始端的是两个启动子，它们的激活不需要任何噬菌体诱导因子，即它们是T4早期启动子。在这些启动子处起始转录会产生几种具有重叠5'端序列的RNA种类，其中最大的估计约15,000个核苷酸长，并且包含该基因簇的所有顺反子。第三个早期启动子位于其中一个顺反子（T4基因47）的蛋白质编码区段内，而至少还有五个启动子位于顺反子间区域，是T4中期启动子，即它们需要T4诱导的DNA结合转录因子MotA。T4基因45 - 44顺反子间区域内一个终止位点处的转录通读对于两种必需的T4 DNA聚合酶（gp43）辅助蛋白gp44和gp62的合成是必需的。相比之下？T4基因43的转录通过跨越regA - 43顺反子间区域内一个终止位点的通读以及位于该终止位点下游的一个MotA依赖型启动子来实现，并且该区域包含一个由T4诱导酶进行加工的位点，该酶也在来自该基因簇的多顺反子mRNA的其他位置进行切割（即基因45.2 mRNA的Shine - Dalgarno序列处）。基因45 - 44和顺反子间区域regA - 43中的终止事件都发生在环段中包含序列5'CUUCGG3'的RNA茎环结构下游。78个碱基对的regA - 43顺反子间区域内的转录终止发生在距离gp43起始密码子AUG约60个核苷酸处，转录起始发生在AUG上游38 - 40个核苷酸处，并且T4依赖型RNA加工发生在转录终止和起始位点之间的几个位点（包括一个GGAG序列）。因此，所有编码gp43的mRNA都包含用于该DNA聚合酶自身抑制的翻译调控序列（相对于AUG为 - 40至 - 1位残基）（安德雷克等人，1988年）。（摘要截断于400字）
