σ^(38)识别的启动子-35区核酸序列特征研究

Character in -35 Region Sequence of Promoter Recognized by σ 38

依据部分资料假定大肠杆菌ＲＮＡ聚合酶的σ３８亚单位在特异启动子的－３５区识别的保守序列是－ＴＣＴＣＣＣ－，合成用其它碱基分别取代这一区域每一个碱基的引物，以ｏｓｍＹ基因片段为模板，通过ＰＣＲ扩增成－３５区含不同碱基序列的转录模板，用体外重组的由σ３８和核心酶（Ｅ）组成的全酶（Ｅσ３８）对这些启动子进行体外转录．结果显示含－ＴＣＴＣＣＣ－序列的启动子的转录水平既低于原始的ｏｓｍＹ启动子，又低于经ＰＣＲ扩增的其它序列．综合研究结果推测，这一区域的保守序列是－ＴＣＴＣＴＣ－．

TCTCCC nucleotide sequence in -35 region of promoter recognized by σ 38 subunit of RNA polymerase in E.coli was supposed to be the conservative.The primers,founding -20 to -60 of osm Y promoter were synthesized.A set of 19 variant promoters that were enriched by PCR was constructed,each carrying a single base substitution within the TCTCCC (nucleotide positions from -39 to -34 relative to the transcription start site of osm Y promoter).Using the DNA plate enriched by PCR and purified E.coli polymerase holoenzyme (Eσ 38 ) in vitro transcription was performed.The following conclusions were drawn from the data presented:1)The nucleotide sequence in -39 to -34 of osm Y promoter was important,and other base substitution might affect its level of in vitro transcription.2)There were rich cytosines in -35 region of promoter recognized by σ 38 .but less or more than three of cytosines might reduce in vitro transcription level.3)The sequence of TCTCCC was not conservative,and TCTCTC was proposed as a consensus sequence of specific promoters recognized by σ 38 .4)The suitable proportion and space structure of two or three dimensions were more important than position of thymidine and cytosine base for σ 38 .