The long term objective of this research is to understand how cells control ribosomal RNA expression in response to changes in the environment. Control of rRNA expression is complex and provides the cell with the proper amount of translational machinery over a broad range of growth rates. In fast growing bacteria, fully half of the cell's transcriptional capacity is devoted to making rRNA, which can reach 80-90 percent of total cellular RNA. Our focus will be on two major topics in rRNA gene expression; characterization of the ribosomal transcriptional antitermination (AT) mechanism and the regulation of rRNA synthesis. We will test models for how AT and termination factors interact with RNA polymerase, first to form an antitermination-transcription complex, then to disassemble this complex at the end of the rrn operon. We will also use strains containing multiply inactivated rrn operons as unique tools to further investigate several current models of rrn expression regulation. The objectives of this proposal are: 1. Identify factors involved in rrn antitermination. Isolate antiterminated transcription complexes and identify the specific factors needed for this process. Use reverse genetics to identify, clone, over-express and purify AT-specific transcription factors and reconstitute this system in vitro. 2. Visualize accessory functions of rrn AT by electron microscopy. Put rrn AT mutations into an identifiable chromosomal operon, and use EM to examine the consequences of disabling this system on rrn transcription. 3. Termination of the AT transcription complex. Manipulate different terminators to investigate what is finally responsible for stopping antiterminated transcription complexes. 4. Role of boxB in rrn AT. Investigate whether this feature of the rrn AT system plays any regulatory role. Mutational analyses of boxB suggest it may bind both an inhibitor and an activator. Search for these postulated activities. 5. Regulation of rrn operon transcription rate. Examine what DNA and protein factors are required for a specific increase in rrn transcription rate under conditions of cellular ribosome insufficiency. 6. PI growth rate-dependent control mutants. Examine known rrn control mutations to determine if they are able to respond to a cellular deficiency of ribosomes. 7. Strain with multiply inactivated operons. Make a strain with a single active rrn operon on a plasmid. Use mutagenesis and gene replacement techniques to do this. 8. Isolation of mutants defective in growth rate-dependent regulation of rRNA synthesis. Use the strain made in #7 to isolate the hypothesized growth rate regulator. This can be accomplished if the remaining operon's expression is under control of a non-regulated promoter. 9. What is the mechanism of rRNA synthesis repression? Use EM to visualize the process of transcriptional down regulation of rRNA genes in the presence of excess plasmid copies of these genes. The importance of this topic lies in the central nature of the translational machinery to other cellular processes.
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