Reeder T, Schleif R
Biology Department, Johns Hopkins University, Baltimore, MD 21218.
J Mol Biol. 1993 May 20;231(2):205-18. doi: 10.1006/jmbi.1993.1276.
At the araBAD promoter, the RNA polymerase-proximal half-site for AraC binding partially overlaps the -35 region. Random and explicit spacing experiments show that both this partial overlapping and AraC binding to the polymerase-proximal half-site are necessary and sufficient for strong transcriptional activation. Normally, this occupancy is generated by the presence of arabinose, which shifts AraC from a DNA looping interaction involving the polymerase-distal half-site and the araO2 site 210 base-pairs away, to an interaction with the two half-sites adjacent to RNA polymerase. Changing the polymerase-proximal half-site to a higher affinity AraC binding site gives activation in the absence of arabinose. Thus, arabinose is not required to transform AraC into an activating conformation. Because the two half-sites of araI are direct repeats, the RNA polymerase proximal and distal surfaces of AraC are not identical. When the araI site was turned around, no spacings were found from which AraC could activate transcription. In light of the strict spacing and orientation requirements for AraC activation, the interactions between AraC and RNA polymerase are likely to be specific and inflexible.
在araBAD启动子处，AraC结合的RNA聚合酶近端半位点部分重叠于 -35区域。随机和明确间距实验表明，这种部分重叠以及AraC与聚合酶近端半位点的结合对于强转录激活而言都是必要且充分的。正常情况下，这种占据是由阿拉伯糖的存在所产生的，阿拉伯糖会使AraC从涉及聚合酶远端半位点和210个碱基对之外的araO2位点的DNA环化相互作用，转变为与RNA聚合酶相邻的两个半位点的相互作用。将聚合酶近端半位点改变为具有更高亲和力的AraC结合位点，在没有阿拉伯糖的情况下也能产生激活作用。因此，将AraC转变为激活构象并不需要阿拉伯糖。由于araI的两个半位点是直接重复序列，AraC的RNA聚合酶近端和远端表面并不相同。当araI位点翻转时，未发现AraC能够激活转录的间距。鉴于AraC激活存在严格的间距和方向要求，AraC与RNA聚合酶之间的相互作用可能是特异性且不灵活的。
