Genetic Switch Controlled by an Unusual Family of Transcription Activators

地球上绝大多数的生物体都是细菌，绝大多数的遗传信息也是细菌。细菌在物种之间以很高的速率交换基因，但人们对这种交换的规则知之甚少，尽管它们对理解微生物进化和生态学很重要。限制修改（RM）制度似乎扮演着核心的“看门人”角色，其调节对其分布和看门人角色都至关重要。大量的RM系统由小的“C蛋白”控制，这些“C蛋白”激活它们自身基因的转录和下游限制性内切酶的转录；在大肠杆菌和芽孢杆菌等不同的细菌中发现了密切相关的C蛋白。这令人惊讶，原因有二。首先，在转录激活剂中实现广泛的宿主范围特别困难，转录激活剂必须与一系列RNA聚合酶进行有效接触。其次，C蛋白比典型的转录激活因子小得多。这些不寻常的激活剂的调控逻辑和作用只得到了有限的表征，但最近确定了C蛋白的结构。通过对显著的C蛋白家族的研究，将提高对转录激活和RM系统功能的理解。将测试四种假设，重点是来自肠杆菌普通变形杆菌（c.p uvi）的C蛋白。第一个假设是，相对于保护性甲基转移酶，对c.p uviii的需求延迟了内切酶基因pvuIIR的表达。这将防止RM系统基因进入新细菌时细胞死亡。第二种假设是靶序列（“C盒”）的不对称调节C蛋白结合并影响其作为调节开关的行为。第三种假说是c.p uvi通过接触RpoD的第4区（sigma70）激活转录，这有助于解释C蛋白异常广泛的宿主范围。第四个假设是c.p uvii和RNA聚合酶全酶是体外激活pvuIICR转录的必要和充分条件，这也有助于解释广泛的宿主范围。更广泛的影响包括研究生培训，包括他们自愿参加教学活动和参加国家或国际会议；对本科生和医学生进行研究培训，并通过出版物、研讨会和提供关于P.I.的文章链接继续传播数据他的学术网站。间接影响将包括微生物遗传学、分子生物学和生物信息学领域的教学；发展生物信息学和蛋白质组学/基因组学的教育和研究项目。

The vast majority of living organisms on earth are bacteria, and the vast majority of genetic information is bacterial. Bacteria exchange genes between species at a high rate, but the rules governing this exchange are poorly understood despite their importance for understanding microbial evolution and ecology. Restriction-modification (RM) systems appear to play a central "gatekeeper" role, and their regulation is crucial to both their distribution and the gatekeeper role. A substantial number of RM systems are controlled by small "C proteins" that activate transcription of their own genes and those of the downstream restriction endonucleases; closely-related C proteins have been found in bacteria as different as E. coli and Bacillus. This is surprising for two reasons. First, broad host range is particularly difficult to achieve among transcriptional activators, which must make productive contacts with a range of RNA polymerases. Second, the C proteins are much smaller than typical transcription activators. The regulatory logic and actions of these unusual activators have received only limited characterization, but the structure for a C protein has recently been determined. The understanding of both transcriptional activation and of RM system function will be improved by studying the remarkable C protein family. Four hypotheses will be tested, focusing on a C protein from the enterobacterium Proteus vulgaris (C.PvuII). First is the hypothesis that the requirement for C.PvuII delays expression of the endonuclease gene pvuIIR relative to that for the protective methyltransferase. This would prevent cell death when the RM system genes move into a new bacterium. Second is the hypothesis that asymmetries in the target sequences ("C boxes") serve to modulate C protein binding and affect its behavior as a regulatory switch. Third is the hypothesis that C.PvuII activates transcription via contact to region 4 of RpoD (sigma70), helping to explain the unusually broad host range of the C proteins. Fourth is the hypothesis that C.PvuII and RNA polymerase holoenzyme are necessary and sufficient for in vitro activation of pvuIICR transcription which, again, would help to explain the broad host range. Broader impacts include graduate student training, including their voluntary participation in teaching activities and attendance at national or international meetings; research training of undergraduates and medical students and continued dissemination of data via publication, seminars, and providing links to articles on the P.I.'s academic web site. Indirect impacts would include teaching in the areas of microbial genetics, molecular biology, and bioinformatics; and development of an educational and research program in bioinformatics and proteomics/genomics.