Isolation, identification, and transcriptional specificity of the heat shock sigma factor sigma32 from Caulobacter crescentus.
Isolation, identification, and transcriptional specificity of the heat shock sigma factor sigma32 from Caulobacter crescentus.
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新月柄杆菌热休克因子 sigma32 的分离、鉴定和转录特异性。
DOI：
10.1128/jb.178.7.2094-2101.1996
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发表时间：
1996
影响因子：
3.2
通讯作者：
Newton,A
中科院分区：
文献类型：
--
作者：
Wu,J;Newton,A
我们报告的鉴定新月柄杆菌热休克因子σ 32作为一个34 kDa的蛋白质，与RNA聚合酶全酶copurifies。根据该蛋白的N端氨基酸序列，设计了一个简并寡核苷酸探针，鉴定了相应的基因rpoH，该基因编码的蛋白质分子量为33，659 Da，其氨基酸序列与已知的大肠杆菌热休克因子相似（41%同一性）、铜绿假单胞菌（40%同一性）和弗氏柠檬酸杆菌（38%同一性）。孤立的C. crescentus基因补充了E. coli rpoH缺失株在37 ℃下进行了表达，Western blot（免疫印迹）分析证实该基因产物与E. coli sigma 32蛋白。纯化的RpoH蛋白在RNA聚合酶核心酶存在下特异性识别C. crescentus dnaK基因，并且在该启动子的-10或-35区域中的碱基对取代废除转录。S1核酸酶图谱表明，在正常生长条件下，rpoH转录物来源于两个启动子，P1和P2。P2启动子类似于sigma 32启动子共有序列，并且P2特异性转录物在热休克期间显著增加，而P1特异性转录物保持相对恒定。这些结果表明，虽然C. crescentus sigma 32与其E. coli的C. crescentus rpoH基因含有一个新的启动子结构，可能在环境胁迫下受到正性自调节。
We report the identification of the Caulobacter crescentus heat shock factor sigma32 as a 34-kDa protein that copurifies with the RNA polymerase holoenzyme. The N-terminal amino acid sequence of this protein was determined and used to design a degenerate oligonucleotide as a probe to identify the corresponding gene, rpoH, which encodes a predicted protein with a molecular mass of 33,659 Da. The amino acid sequence of this protein is similar to those of known bacterial heat shock sigma factors of Escherichia coli (41% identity), Pseudomonas aeruginosa (40% identity), and Citrobacter freundii (38% identity). The isolated C. crescentus gene complements the growth defect of an E. coli rpoH deletion strain at 37 degrees C, and Western blot (immunoblot) analysis confirmed that the gene product is related to the E. coli sigma32 protein. The purified RpoH protein in the presence of RNA polymerase core enzyme specifically recognizes the heat shock-regulated promoter P1 of the C. crescentus dnaK gene, and base pair substitutions in either the -10 or -35 region of this promoter abolish transcription. S1 nuclease mapping indicates that rpoH transcripts originate from two promoters, P1 and P2, under the normal growth conditions. The P2 promoter is similar to the sigma32 promoter consensus, and the P2-specific transcript increases dramatically during heat shock, while the P1-specific transcript remains relatively constant. These results suggest that although the structure and function of C. crescentus sigma32 appear to be very similar to those of its E. coli counterpart, the C. crescentus rpoH gene contains a novel promoter structure and may be positively autoregulated in response to environmental stress.
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影响因子：
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作者：
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影响因子：
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作者：
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DOI：
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发表时间：
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影响因子：
11.1
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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