STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE PROMOTER REGION INVOLVED IN FULL EXPRESSION OF THE CRYIIIA TOXIN GENE OF BACILLUS-THURINGIENSIS
STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE PROMOTER REGION INVOLVED IN FULL EXPRESSION OF THE CRYIIIA TOXIN GENE OF BACILLUS-THURINGIENSIS
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DOI：
10.1111/j.1365-2958.1994.tb00405.x
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发表时间：
1994-07-01
影响因子：
3.6
通讯作者：
LERECLUS, D
中科院分区：
文献类型：
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作者：
AGAISSE, H;LERECLUS, D
苏云金芽孢杆菌cryyiiia毒素基因的启动子区域至少由三个结构域组成：上游区域从核苷酸位置-635延伸至-553（参考cryyiiia的翻译起始密码子），内部区域从核苷酸位置-553延伸至-367，下游区域从核苷酸位置-367延伸至+18。缺失分析和对lacZ基因的转录融合表明，cryIIIA的完全表达需要上游和下游区域的关联。引物延伸实验发现一个主要的cryiia转录物（命名为T-129）开始于核苷酸位置-129，另一个转录物（命名为T-558）开始于核苷酸位置-558。负责T-558起始的启动子-35区域的突变表明，上游启动子对于cryIIIA的完全表达是必需的，尽管不是充分的。携带上述cryIIIA启动子的DNA区域的删除不会影响cryIIIA的完全表达，也不会修饰T-129的5'端。综上所述，这些结果表明T-129的5'端不是转录起始位点。因此，我们认为T-129是由上游启动子（T-558）启动的mRNA加工产生的，在核苷酸位置-129上产生了一个5'末端的稳定mRNA。从引物延伸分析和转录融合到lacZ，上游启动子似乎在营养生长阶段弱但显著表达，在孢子形成开始时被激活，并至少保持活性直到t(5)。然而，与其他哭泣基因的启动子不同，该启动子类似于sigma(A)依赖性启动子，而不是孢子特异性启动子。因此，该启动子可能被E sigma(A)形式的RNA聚合酶转录。在孢子形成开始时的激活可能是由于抑制因子的消失，或固定阶段特异性激活因子的出现。
The promoter region of the cryIIIA toxin gene of Bacillus thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions -635 to -553 (with reference to the translational start codon of cryIIIA), an internal region extending from nucleotide positions -553 to -367, and a downstream region extending from nucleotide position -367 to +18. Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region. Primer extension experiments reveal a major cryIIIA transcript (designated T-129) starting at nucleotide position -129 and another transcript (designated T-558) starting at nucleotide position -558. Mutation in the -35 region of the promoter responsible for the initiation of T-558 indicates that the upstream promoter is essential for full expression of cryIIIA, although not sufficient. Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5' end of T-129. Taken together, these results indicate that the 5' end of T-129 is not a transcriptional start site. Therefore, we propose that T-129 results from the processing of the mRNA initiated at the upstream promoter (T-558), generating a stable mRNA with a 5' extremity at nucleotide position -129. From primer extension analysis and transcriptional fusions to lacZ, it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporulation and remains active at least until t(5). However, unlike the promoters of other cry genes, this promoter is similar to sigma(A)-dependent promoters rather than sporulation-specific promoters. This promoter may therefore be transcribed by the E sigma(A) form of RNA polymerase. Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary-phase-specific activator.