The regulation of gene transcription is one of the most important mechanisms used by cells to respond to alterations in their internal or external environment. To ensure a rapid response, transcriptional regulators are often synthesized and stored in a dormant form, becoming active only in response to a specific signal. Much of the biological interest in transcriptional responses focuses on how the activity of transcription factors is regulated. Sequestration of transcription factors away from their DNA targets, by temporary attachment to cellular membranes, is emerging as an elegant mechanism to regulate their activities. Several membrane-anchored transcription factors (MTFs) have been experimentally identified whose access to the transcriptional machinery is regulated by proteolytic cleavage from the membrane. This process is broadly referred to as regulated intramembrane proteolysis (RIP). RIP is a newly discovered mechanism that controls many important signaling pathways conserved from bacteria to humans. The discovery of an essential role for RIP in biologically relevant processes makes it important to understand the full gamut of RIP function. However, RIP is little understood and so far there has been no systematic study of RIP in any organism. This project is for a genome-wide study of RIP in Anabaena variabilis 29413, a multicellular cyanobacterium, capable of simultaneously carrying out agriculturally relevant O2-producing photosynthesis and O2-labile N2-fixation, as well as bio-solar H2 gas production. In addition, its vegetative cells can differentiate into three other types of cells in response to environmental changes, which presents a rare opportunity to investigate gene regulation of several differentiation processes in a single bacterium. Given its unique capabilities, A. variabilis provides a very attractive experimental system to study fundamental biological problems, especially for agriculture and bio-energy related problems. An analysis of the genomic sequence of A. variabilis identified 19 putative RIP genes that encode 5 RIP proteases and 14 MTFs. Literature surveys imply that some orthologs of the putative RIP genes may be involved in regulation of stress responses, chloroplast development, cellular differentiation, cell-cell communication, and photosynthesis, as well as aerobic N2-fixation. Therefore, the 19 putative RIP genes in A. variabilis may play important biological roles. The aims of the project are 1) to obtain clues for the cellular functions of 19 putative RIP genes by inactivating these genes; 2) to determine the expression patterns of these genes for further definition of their functions during A. variabilis growth and development, and in response to various stress conditions; 3) to determine the proteolytic activities of 5 putative RIP proteases in a reconstituted E. coli system; and 4) to identify specific MTF/RIP protease pairs.
Broader Impact The PI has a history of mentoring students and in this project will broaden participation through outreach activities in an established collaboration at Michigan State University with a minority female professor who participates in the Charles Drew program. The Charles Drew Program is a retention and academic enhancement program for high-achieving undergraduate students and graduates from underrepresented minority groups. The PI will mentor students in the program, provide career development guidance to the participants, and serve as a research advisor to interested students. In this project the PI will provide hands-on research experiences to four undergraduates.
