In Vivo Studies of Abortive Initiation

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DESCRIPTION (provided by applicant): Transcription is the first step in gene expression and the step at which most gene regulation occurs. Transcription in all cells is carried out by multi-subunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans. Whereas initiation of DNA synthesis by DNA polymerase requires use of a primer, it is widely accepted that the initiation of RNA synthesis by RNAP occurs "de novo" (i.e. RNAP initiates RNA synthesis using free NTPs only). With the proposed research we seek to challenge this conventional paradigm. Specifically, we will investigate the hypothesis that a significant fraction of transcription does not occur "de novo", but rather relies upon use of small ~2-5 nt RNA transcripts, "nanoRNAs", that influence gene expression by serving as primers to initiate transcription. The first aim will use microarray analysis, chromatin immunoprecipitation (ChIP) coupled with fully tiled high density DNA microarrays (ChIP-on-chip), and high-throughput sequencing to define how nanoRNA- dependent priming can influence gene expression. The second aim will use high-throughput sequencing to identify those nanoRNAs that function to prime transcription initiation in vivo. The third aim will use microarray analysis, ChIP-on-chip, and high-throughput sequencing to determine the extent to which nanoRNA-dependent priming impacts gene expression during physiological growth conditions. The proposed research is part a long-range effort to elucidate fundamental mechanisms of gene regulation in bacteria. These studies have the potential to redefine our view of a fundamental process that occurs in all living cells (i.e. transcription) and, in parallel, uncover a novel class of regulatory small RNAs, "nanoRNAs", that function in all living cells via a novel mode of action.