The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms. The regulation of the synthesis of the translation machinery in response to the nutritional state of the cell has been a central issue in the study of microbial physiology for at least fifty years. More recently, it has also become clear that an understanding of the mechanisms responsible for rRNA transcription in Escherichia coli can provide fundamental insights into understanding mechanisms of transcription in general. We are poised to address questions central to our understanding of general transcription mechanisms and to our understanding of how different regulatory systems work together. The questions addressed in the proposal are divided into four specific aims in arbitrary order. In the first aim, we will study an unanticipated role of the C-terminal domain of the alpha subunit of RNA polymerase in bacterial promoter recognition. In the second aim, we propose to determine how the rrn transcription factor Fis increases transcription from sites far upstream of the -35 element and how it affects different steps in the transcription mechanism. In the third aim, we propose to determine when different mechanisms contribute to regulation of the rrn P1 and rrn P2 promoters in response to changing nutritional conditions and how this maintains homeostasis. Also as part of this aim, we will determine the molecular interactions responsible for a key determinant in the control of rRNA transcription, the lifetime of the open complex, we will examine a new, previously unrecognized regulator of rRNA transcription, and we will begin a genomic study of tRNA promoters. In the fourth specific aim, we will use the tools of cell biology to determine the locations of rRNA operons in the bacterial nucleoid.
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