Tsui H C, Winkler M E
Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, 77030, USA.
Biochimie. 1994;76(12):1168-77. doi: 10.1016/0300-9084(94)90046-9.
The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA. In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.
大肠杆菌的复杂amiB - mutL - miaA - hfq - hflX - hflK - hflC超级操纵子包含了几个基本细胞过程的重要基因，包括细胞壁水解（amiB）、DNA修复（mutL）、tRNA修饰（miaA）和蛋白水解（hflX - hflK - hflC）。我们在此报告该超级操纵子中mutL、miaA和hfq基因的转录模式及可能的转录后调控。对从细菌染色体转录的mRNA进行核糖核酸酶保护分析表明，mutL和miaA是共转录的。此外，还鉴定出两个内部启动子PmiaA和P1hfq，并将它们定位在各自翻译起始位点上游201和837个核苷酸处。PmiaA与σ70 RNA聚合酶共有序列的 - 10和 - 35区域的匹配较差，但它包含多个潜在的Fis结合位点以及一个带有多聚（A）序列的富含AT的上游区域。Fis结合位点后接富含AT区域的基本排列方式与rRNA操纵子以及一些tRNA和tRNA修饰基因的启动子相同。作为对mutL和miaA调控初步研究的一部分，我们测量了缺乏核糖核酸酶E、核糖核酸酶III或两者的同基因rne、rnc和rne rnc双突变体中的转录本量。与rne +亲本相比，rne3071突变体中稳态水平的mutL - miaA共转录本、PmiaA转录本和P1hfq转录本的量分别增加了8倍、9倍和3倍。相反，rnc105突变体及其rnc +亲本中这三种转录本的量相同。这些结果表明，mutL、miaA和hfq的表达可能受多种机制调控，包括上游基因的共转录程度、内部启动子强度的调节以及核糖核酸酶E的活性。本文提出了一个核糖核酸酶E介导的转录后调控模型，该模型可能在不同生长条件下，特别是在营养物质上调期间，协调mutL表达与复制以及miaA表达与tRNA量之间的关系。
