A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis.

Taking advantage of the known sequence conservation of portions of bacterial sigma factor proteins, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction (PCR) to amplify DNA sequences from the chlamydial genome. The PCR products were used as a probe to recover the genomic fragments from a library of cloned murine Chlamydia trachomatis DNA. Sequence analysis of one of these clones revealed striking homology to the sigma-70 protein of Escherichia coli and the sigma-43 protein of Bacillus subtilis, strongly implying that this locus (sigA) encodes the major vegetative sigma factor of murine C. trachomatis. This PCR-based approach will be broadly applicable to the cloning of major sigma factors from other eubacteria.     

Sigma54依赖性转录起始

作为细菌RNA聚合酶(RNAP)的组成型辅助因子,sigma70和sigma54分别参与了原核细胞不同类型基因的转录起始调控。sigma70负责管家基因的自发转录起始;而sigma54负责应激信号相关的基因转录起始。sigma54与RNAP形成复合物后,会通过空间阻滞的方式阻碍DNA进入RNAP中,抑制基因转录起始。当细胞环境变化后,特定应激信号会通过细菌增强子结合蛋白(bEBP)诱发sigma54的构象发生变化,解除sigma54对RNAP的抑制,启动sigma54依赖的基因转录。最近的结构生物学研究揭示了sigma54依赖性转录起始的若干复合物结构,包括全酶、封闭式复合物、2个中间状态复合物及开放式复合物。通过分析这些转录起始复合物的结构,本文阐述了转录起始过程中复合物的结构变化。描述并分析了sigma54和bEBP在转录起始过程中所发挥的功能。本文有助于了解转录起始分子水平的变化,为深入理解sigma54和bEBP促进转录起始的分子机制提供了参考。