Passador L, Linn T
Department of Microbiology and Immunology, Faculty of Medicine, University of Western Ontario, London, Canada.
J Bacteriol. 1992 Nov;174(22):7174-9. doi: 10.1128/jb.174.22.7174-7179.1992.
In order to delineate the region involved in feedback regulation of the RNA polymerase beta subunit (encoded by rpoB), a collection of rpoB-lacZ translational fusions with different endpoints both upstream and downstream of the rpoB start site was assembled on lambda phage vectors. The extent of translational repression of beta was monitored by measuring beta-galactosidase levels in monolysogens of the fusions under conditions of increased intracellular concentrations of beta and beta' achieved via the induction of rpoBC expression from a multicopy plasmid. A construct containing as little as 29 bp upstream of the start of rpoB exhibited repression of beta-galactosidase activity to the same extent as a construct encoding the full upstream region. A construct which carried only 70 bp of the rpoB structural gene exhibited very little repression, while constructs which carried 126 or 221 bp of rpoB exhibited approximately the same degree of repression as a construct which carried 403 bp. These data suggest that the sequences important for feedback regulation of beta translation extend more than 70 bp into rpoB but are completely contained within a region which spans the sequences from 29 bp upstream to 126 bp downstream of the translational start site.
为了描绘参与RNA聚合酶β亚基(由rpoB编码)反馈调节的区域,在λ噬菌体载体上构建了一系列rpoB-lacZ翻译融合体,这些融合体在rpoB起始位点的上游和下游具有不同的端点。通过在多拷贝质粒诱导rpoBC表达从而增加细胞内β和β'浓度的条件下,测量融合体单溶原菌中的β-半乳糖苷酶水平,来监测β的翻译抑制程度。一个在rpoB起始位点上游仅含29 bp的构建体,其β-半乳糖苷酶活性的抑制程度与编码完整上游区域的构建体相同。一个仅携带rpoB结构基因70 bp的构建体几乎没有抑制作用,而携带rpoB 126或221 bp的构建体表现出与携带403 bp的构建体大致相同程度的抑制作用。这些数据表明,对β翻译进行反馈调节的重要序列延伸到rpoB内超过70 bp,但完全包含在一个从翻译起始位点上游29 bp到下游126 bp的区域内。