Cunningham Louise, Guest John R
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of SheffieldWestern Bank, Sheffield S10 2TNUK.
Microbiology (Reading). 1998 Aug;144 ( Pt 8):2113-2123. doi: 10.1099/00221287-144-8-2113.
The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16.3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; lpdA) is the distal gene of another cluster containing two promoters located at 2.7 min: Ppdh pdhR-aceEF-Plpd lpdA. The responses of the suc and lpd promoters to different environmental conditions and to regulator defects were investigated with appropriate lacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with lpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by sigma 38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the lpd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or sigma 38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the lpd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc. Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.
编码琥珀酸脱氢酶（sdhCDAB）、2-氧代戊二酸脱氢酶复合体（ODH，E1o和E2o；sucAB）的特定组分以及琥珀酰辅酶A合成酶（sucCD）的基因在大肠杆菌染色体上16.3分钟处形成一个含有两个启动子的簇：Psdh sdhCDAB - Psuc sucAB - sucCD。编码2-氧代戊二酸和丙酮酸脱氢酶复合体的硫辛酰胺脱氢酶组分（E3；lpdA）的基因是另一个位于2.7分钟处含有两个启动子的簇的远端基因：Ppdh pdhR - aceEF - Plpd lpdA。利用合适的lacZ融合体研究了suc和lpd启动子对不同环境条件和调节因子缺陷的反应，以了解sucAB基因的表达如何与sdhCDAB - sucABCD簇中的其他基因以及lpdA的表达共同调节。suc启动子的表达受到整合宿主因子（IHF）的抑制，并被σ38部分激活，但不受ArcA、FNR、CRP、FruR或Fis的调节，也不受葡萄糖或厌氧条件的抑制，这表明已确立的ODH合成的分解代谢物和厌氧抑制作用是在其他地方施加的。相反，lpd启动子受到葡萄糖（通过一种不依赖CRP的机制）和厌氧条件（由ArcA介导）的抑制，并被Fis激活，但不受FNR、FruR、IHF或σ38的调节。这些观察结果支持这样一种观点，即sucABCD基因的转录主要在上游的sdh启动子处起始和调节，并且lpd启动子是与Psdh独立共同调节（主要通过ArcA介导的抑制）而不是与Psuc共同调节。通过在rnc和rne突变体中检测到一个10 kb的转录本，但在亲本菌株中未检测到，从而获得了从Psdh对整个sdhCDAB - sucABCD区域进行共转录的直接证据。在sdhCDAB - sucABCD基因间区域鉴定出三个RNaseIII特异性加工位点，这些位点导致通读转录本的极端不稳定性。通过解释在Northern印迹中观察到的转录本亚片段模式来定位核酸内切酶加工的其他位点。
