A mutant spacer sequence between-35 and-10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent
A mutant spacer sequence between-35 and-10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent
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DOI：
10.1073/pnas.0401929101
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发表时间：
2004-05-04
影响因子：
11.1
通讯作者：
Adhya, S
中科院分区：
文献类型：
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作者：
Liu, MF;Tolstorukov, M;Adhya, S
为了确定-35和-10元件之间的间隔区是否发挥任何序列特异性作用，我们将cAMP依赖性lac启动子的间隔区内的富含GC的序列（（-20）CCGGCTCG（-13））随机化，并选择了一种激活剂非依赖性突变体，其显示出非常高的内在活性。超活性启动子是通过在间隔区内掺入特定的10 bp长的富含AT的DNA序列获得的，称为-15序列，其必须与超活性的-10序列的上游端并置。转录增强仅在-35元件存在时起作用。间隔区序列增强RNA聚合酶结合和开放复合物形成。在lac启动子中分离，当置于另外两个不相关的启动子时，它也增强了转录。序列分析显示在-18/-9区域中低GC含量和立体化学柔性TG：CA和TA：TA二聚体步骤的丰度，以及在该区域中柔性二聚体步骤的存在与启动子的内在强度之间的强相关性。
To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.