ROLE OF THE SIGMA-70-SUBUNIT OF RNA-POLYMERASE IN TRANSCRIPTIONAL ACTIVATION BY ACTIVATOR PROTEIN PHOB IN ESCHERICHIA-COLI
ROLE OF THE SIGMA-70-SUBUNIT OF RNA-POLYMERASE IN TRANSCRIPTIONAL ACTIVATION BY ACTIVATOR PROTEIN PHOB IN ESCHERICHIA-COLI
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DOI：
10.1101/gad.7.1.149
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发表时间：
1993-01-01
影响因子：
10.5
通讯作者：
SHINAGAWA, H
中科院分区：
文献类型：
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作者：
MAKINO, K;AMEMURA, M;SHINAGAWA, H
在大肠杆菌中，磷酸盐饥饿诱导的磷酸盐（pho）调节子基因的转录，除了含有主要σ-因子σ 70的RNA聚合酶全酶外，还需要特异性激活蛋白PhoB。为了研究转录激活的机制，并确定参与与PhoB特异性相互作用的RNA聚合酶的亚基，我们试图分离特异性缺陷的pho基因表达的rpoA和rpoD突变体。我们分离出两个具有这种特性的rpoD突变体，但没有具有类似特性的rpoA突变体。rpoD突变改变了simga 70羧基末端区域中假定的螺旋-转角-螺旋（HTH）基序的第一螺旋内和附近的氨基酸。在这些突变体中，PHO启动子在体内的活动严重减少，而PHOB独立启动子的活动最多只受到轻微的影响。重组的突变体RNA聚合酶全酶在转录pstS基因（pho基因之一）方面存在严重缺陷，而它们在转录PhoB独立启动子方面是有效的。磷酸化的PhoB，它结合到具有高亲和力的pho启动子，介导的特异性结合的野生型全酶的pstS启动子，但它不介导的突变体全酶的结合。这些结果表明，PhoB促进RNA聚合酶和pho启动子之间的特异性相互作用的转录激活，和第一螺旋的推定HTH基序在相互作用中起着至关重要的作用，可能是通过直接接触与PhoB。
Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major sigma-factor sigma70. To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of simga70. Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.