Abstract
The transition from the formation of the RNA polymerase (RNAP)-promoter open complex step to the productive elongation complex step involves "promoter escape" of RNAP. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two-component σ factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, which mimics σR4, is released gradually as transcription elongation proceeds, whereas YvrHa, which mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP.
Highlights
The proposed model for promoter escape predicts the destabilization of interactions of region 4 with RNA polymerase and DNA
Our results show that the amount of YvrI relative to RNA polymerase (RNAP) is gradually decreased in the elongation complexes as elongation proceeds, whereas the amount of YvrHa relative to RNAP remains constant in the elongation complexes (Fig. 6)
Because a certain amount of YvrI is present in the early elongation complexes, our result is consistent with previous observations that shows a significant fraction of 70 remains associated with RNAP in the early elongation complex [6, 12]
Summary
The proposed model for promoter escape predicts the destabilization of interactions of region 4 with RNA polymerase and DNA. Results: Using a two-component factor, we show that YvrI, mimicking the region 4, is released, whereas YvrHa, mimicking region 2, is retained after promoter escape. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between R4 and RNAP and R4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of to RNAP and stochastic release of . We study the promoter escape model using a two-component factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of R4 and R2 in a RNAP-promoter complex. Our result validates the proposed model for promoter escape and suggests that promoter escape involves little or no change in the interaction of R2 with RNAP
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