Gene expression in bacteria depends critically on the sigma subunit of RNA polymerase, which combines with the core enzyme to form the holoenzyme. The proposed research is aimed at examining functionally important interactions of sigma with the core, with transcription regulators, and with DNA. Genetic tools will be developed to facilitate this analysis in the context of recently determined core and holoenzyme structures. Sigma mediates both promoter recognition and formation of the initiation-competent open (melted) complex through direct contact with conserved promoter elements. To carry out these functions sigma makes critical interactions with the core enzyme, which is highly conserved from bacteria to man. One goal of the proposed research is to use recently developed genetic tools to further examine one such contact: the recently uncovered interaction between the primary sigma factor (sigma 70) and a conserved domain of the beta subunit. This interaction is required for RNAP to recognize the major class of sigma 70-dependent promoters. Sigma 70 has two DNA-binding domains (conserved regions 2 and 4) that make sequence-specific contacts with two discrete promoter elements. Another goal of the proposed research is to further develop genetic assays to facilitate the dissection of these protein-DNA interactions. In particular, region 2 of sigma 70, which mediates promoter-melting, has the ability to bind both double- and single-stranded DNA. Approaches will be developed to identify critical determinants of these two activities. Finally, recent work has indicated that the functions of sigma 70 are not limited to the initiation phase of transcription, but relatively little is known about these additional roles of sigma 70. Studies of the bacteriophage lambda late promoter have revealed the presence of sigma 70 in an early elongation complex, and this lambda system has provided a model for studying the functions of sigma 70 in early elongation. The proposed research addresses several mechanistic questions raised by this work, as well as investigating the extent to which this lambda system reveals general features of the early elongation complex.
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