Characterization Of Novel Dna Elements Necessary For Sigma-Dependent Promoter Proximal Transcriptional Pausing

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Transcription is not necessarily uniform in rate, but instead consists of multiple periods of continuous elongation by RNA polymerase (RNAP) interrupted by occasional pausing events. These pauses have several determined origins, including the interaction of protein co-factors, the influence of transcribed RNA secondary structure, and a direct effect of the sequence composition of the DNA being transcribed. Promoter proximal pauses, in which RNAP pauses tens of nucleotides downstream of the transcription start site, have been observed in systems ranging from bacteria to more complex eukaryotes such as D. melanogaster and humans, and play important regulatory roles in organisms of all levels. In E. coli, [sigma]70-dependent promoter proximal pauses are of particular interest because lambdoid phage late gene promoters require these pauses for the loading of the anti-terminator Q protein, which is necessary for transcription of the phage late genes during lytic growth. The primary interaction that induces [sigma]70-dependent pauses is between the non-template strand of the -10-like sequence and [sigma]70 region 2. However, recent evidence identifies other important sequences including several nucleotides around the pause site itself that are required for pausing (Perdue and Roberts 2010). Mutational studies of lambdoid phage 82 promoter pR' show that a 3-bp GC-rich sequence primarily promotes pausing through the template DNA strand, but also acts through a non-template DNA strand interaction with [sigma]70. I show that this template strand sequence determines where the pause occurs. It is likely that in [sigma]-dependent promoterproximal pausing, the [sigma]70/DNA interaction anchors the elongation complex upstream of the pause site, requiring it to "scrunch" (by analogy to scrunching during initial transcription (Kapanidis et al. 2006)) and to create an enlarged DNA bubble as the active center reaches the pause site. "Scrunched" complexes are energetically strained, but stable at the pause site; my data suggest that the G/C-rich template sequence is a critical element in stabilizing the paused, scrunched structure. Mutational studies of the A/T-rich region demonstrate that the terminal nucleotide of the pause site plays an important role in pause formation, and that the base composition of the A/T-rich sequence determines the likelihood that a transcription complex escapes the pause. My data also suggest that a [sigma]70-dependent paused transcription complex enters the paused state through a pretranslocated state of the enzyme. Through these studies, I have expanded the current understanding of the protein/nucleotide and nucleotide/nucleotide interactions that constitute the [sigma]70dependent promoter proximal paused complex.

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Transcription; Pausing; Phage


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Roberts, Jeffrey Warren

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Lis, John T
Alani, Eric E

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Ph. D., Biochemistry

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Doctor of Philosophy

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dissertation or thesis

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