Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.
Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.
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普瓦泽基立克次体主要大分子合成操纵子的转录特征。
DOI：
10.1128/jb.179.20.6448-6452.1997
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发表时间：
1997
影响因子：
3.2
通讯作者：
Wood,DO
中科院分区：
文献类型：
--
作者：
Shaw,EI;Marks,GL;Winkler,HH;Wood,DO
最近的研究表明，普氏立克次体可以在起始水平上调控特定基因的转录。然而，很少有关于操纵子的存在和协调的基因调控在这个专性细胞内寄生菌的信息。为了解决这些问题，我们集中在rpoD基因连锁群（greA-开放阅读框架23 [ORF23]-dnaG-rpoD），其中包括主要大分子合成操纵子（MMSO）的立克次体类似物（ORF23-dnaG-rpoD）。立克次体MMSO由编码未知功能蛋白的ORF、DNA引发酶的结构基因（dnaG）和RNA聚合酶的主要σ因子（rpoD）组成。使用RNA酶保护测定（RPA）来确定这些基因是否被组织成由多个启动子控制的操纵子以及这些基因相对于彼此产生的转录物的量。RPA与探针跨越270个碱基的greA-ORF 23插入区确定了一个假定的转录启动子内的插入序列。跨越连锁群接下来的4，041个碱基的多个RPA探针证明了连续转录物的存在，因此存在操纵子。跨越dnaG-rpoD区域的探针显示，另外两个mRNA片段也受到保护，这使我们能够确定dnaG内rpoD的其他推定启动子。引物延伸确定三种转录物的5 '端分别由腺嘌呤（位于ORF 23上游227个碱基）和尿嘧啶和腺嘌呤（分别位于rpoD上游336和250个碱基）组成。对三个ORF产生的转录物的定量确定转录物的相对量（ORF 23对dnaG对rpoD）为1：2.7：5.1。
Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.