Bacterial Functions Involved in Cell Growth Control

哥德斯曼 项目名称摘要 我们以大肠杆菌为模型系统，继续研究依赖能量的蛋白质降解在调节基因表达中所起的作用。RSSB是一种调节固定相sigma因子rpos降解的蛋白质，已被发现向ClpXP蛋白酶递送rpos。降解是通过RSSB的磷酸化来表示的；我们正在研究体内和体外的磷酸化和去磷酸化的机制。对RSSB基因的缺失分析表明，蛋白质的N端和C端都是降解rpos所必需的。C末端的缺失导致底物以一种不活跃但稳定的形式被隔离。被删除的衍生物ClpX和rpos之间的特定相互作用将在体外进行研究。 除了调节RPOS的降解外，RPOS的翻译还受到两个小RNAs的正向调节，DSRA和RPRA。这些小RNA中的每一个区域都可以与rpos前导mRNA配对。这打乱了RPOS领导者的二级结构，允许进行翻译。DsrA的启动子受温度调节(低温开启，高温关闭)。我们发现，温度调节可以通过启动子-10区域的单个碱基对变化来完全解除，这表明不寻常的启动子结构可能介导了温度调节。RPRA被认为是dsrA突变体的抑制因子，受RCSC和RCSB两个组分调节。这些调节剂还可以促进被膜多糖的合成，并上调细胞分裂蛋白；它们被细胞表面的压力激活。当它们被激活时，RPOS合成以RPRA依赖的方式增加。通过对这两个小RNA的比较，可以开发出对RPOS进行积极调控所需的共识序列，并展示了如何感知多种环境信号来改变RPOS的可用性。 NICHD的Gisela Storz博士和Affymetrix的Carsten Rosenow博士的实验室最近完成了一项合作，以识别大肠杆菌中的其他小RNA。我们确定了保守的基因间隔区，并用微阵列分析和Northern blotts扫描了其中的一些区域，以确定17个新的小RNA(SRNAs)和5个编码短ORF的新mRNAs。此外，我们还发现许多sRNA与RNA结合蛋白Hfq结合，Hfq是DSRA作用和rpos翻译所必需的。我们的方法可以应用于在其他生物体中寻找小RNA。新发现的sRNAs之一，RyhB，负调控SDH的翻译，编码琥珀酸脱氢酶。RyhB的合成反过来又受到铁依赖的毛皮抑制因子的负调控。当铁受到限制时，细胞下调SDH，可能允许原本用于组装琥珀酸脱氢酶的铁可用于必要的功能。其他新的sRNA对rpos有正向和负向的调节作用，而许多sRNA的功能尚不清楚。

Gottesman Project Title Summary We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia colias a model system. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to present RpoS to the ClpXP protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. Deletion analysis of the rssB gene demonstrates that both the N-terminus and C-terminus of the protein are necessary for degradation of RpoS. Deletions of the C-terminus lead to sequestration of substrate in an inactive but stable form. Specific interactions between the deleted derivatives, ClpX, and RpoS will be studied in vitro. In addition to regulation of RpoS degradation, RpoS translation is positively regulated by two small RNAs, DsrA and RprA. Regions within each of these small RNAs can pair with the RpoS leader mRNA. This disrupts the secondary structure of the RpoS leader, allowing translation. The promoter of dsrAis regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation can be fully relieved by a single base pair change in the -10 region of the promoter, suggesting that unusual promoter structures may be mediating temperature regulation. RprA, identified as a suppressor of dsrAmutants, is regulated by the two component RcsC and RcsB regulators. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein; they are activated by cell surface stress. When they are activated, RpoS synthesis increases in an RprA-dependent fashion. Comparison between the two small RNAs allows development of a consensus sequence necessary for positive regulation of RpoS, as well as demonstrating how multiple environmental signals can be sensed to modify RpoS availability. A collaboration with the laboratory of Dr. Gisela Storz in NICHD and Dr. Carsten Rosenow at Affymetrix to identify other small RNAs in E. coli was recently completed. We identified conserved intergenic regions and scanned a number of these both with microarray analysis and Northern blots to identify 17 novel small RNAs (sRNAs) and five novel mRNAs encoding short ORFs. In addition, we found that many of these sRNAs bind to the RNA binding protein, Hfq, essential for DsrA action and RpoS translation. Our approaches can be applied to searches for small RNAs in other organisms. One of the newly identified sRNAs, RyhB, negatively regulates translation of sdh, encoding succinate dehydrogenase. RyhB synthesis in turn is negatively regulated by the Fe-dependent Fur repressor. When iron is limiting, the cell down-regulates sdh, possibly allowing Fe that would otherwise be used in assembling the succinate dehydrogenase enzyme to be available for essential functions. Other novel sRNAs positively and negatively regulate RpoS, while the function of many is not yet known.