BACTERIAL FUNCTION INVOLVED IN CELL GROWTH CONTROL

我们继续研究依赖能量的蛋白质所扮演的角色 利用大肠杆菌，降解在调节基因表达中发挥作用 作为一个模范系统。RSSB，一种调节细胞降解的蛋白质 已发现固定相西格玛因子rpos可调节rpos。 活性和降解性。提取高产的RSSB，但不 那些缺乏RSSB的人能够刺激依赖ClpXP的 RPO的降解，允许开发一种用于 纯化，以导致对刺激因素的详细研究 退化。RPOS翻译由DSRA管理，DSRA是一个小型稳定的85 核苷酸核糖核酸。突变分析表明，位于 RNA的5‘末端是调节rpos所必需的，并显示 与RPOS未翻译的前导序列的一个区域同源 牵涉到翻译法规中。这表明配对 DSRA RNA和RPOS前导之间打开了前导结构， 允许翻译。DSRA的第二项活动，克服 沉默与类核相关的HNS蛋白，是 独立于第一个茎环，表明该RNA有两个 不同的活动。我们已经使用了Ci-HNS杂合蛋白，其中 对噬菌体lambda的免疫依赖于噬菌体之间的寡聚作用 HNS域，以证明反沉默是通过破坏HNS来实现的 齐聚作用。DSRA和其他小型RNA正在获得越来越多的 识别为新的调节分子。 在对lambda Xis蛋白降解的研究中，我们发现 它容易被两个不同的依赖于ATP的大肠杆菌降解 蛋白酶、Lon和FtsH。感染病毒的快速降解有助于确保快速和 由lambda稳定的溶原化。在对Lon的诱变研究中 蛋白酶，我们发现C-末端的缺失和点突变 移除或灭活蛋白分解活性部位的结构域仍然存在 当产量过剩时，能够部分补充离子突变体。此时将显示以下内容 这是因为底物结合结构域保留在N-末端 缺失的蛋白质中，底物的结合足以 干扰底物功能，模仿降解的效果。 这将允许分析底物识别和结合 Lon蛋白水解酶的研究不依赖于蛋白质的降解。

We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia coli as a model system. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to regulate RpoS activity as well as degradation. Extracts overproducing RssB but not those devoid of RssB are able to stimulate the ClpXP-dependent degradation of RpoS, allowing the development of an assay for purification, to lead to detailed study of the factors that stimulate degradation. RpoS translation is regulated by DsrA, a small stable 85 nucleotide RNA. Mutational analysis indicates that a stem-loop at the 5' end of the RNA is necessary for regulation of RpoS, and shows homology to a region of the untranslated leader of RpoS that has been implicated in translational regulation. This suggests that pairing between the DsrA RNA and the RpoS leader opens up the leader structure, allowing translation. The second activity of DsrA, overcoming the silencing associated with the nucleoid associated HNS protein, is independent of the first stem-loop, demonstrating that this RNA has two distinct activities. We have used cI-HNS hybrid proteins, in which immunity to bacteriophage lambda depends on oligomerization between the HNS domains, to demonstrate that anti-silencing works by disrupting HNS oligomerization. DsrA and other small RNAs are gaining increasing recognition as novel regulatory molecules. In studies on the degradation of the lambda Xis protein, we find that it is subject to degradation by two distinct E. coli ATP-dependent proteases, Lon and FtsH. Rapid degradation of Xis helps insure rapid and stable lysogenization by lambda. In mutagenesis studies of the Lon protease, we find that deletions and point mutations in the C-terminal domain that remove or inactivate the proteolytic active site are still able to partially complement lon mutants when overproduced. This appears to be because the substrate binding domain is retained in the N-terminus of the deleted proteins, and binding of substrate is sufficient to interfere with substrate function, mimicking the effect of degradation. This will allow the analysis of substrate recognition and binding by the Lon protease to be studied independently of protein degradation.