In a round of transcription, RNA polymerase goes through three phases -- initiation, elongation, and termination -- to synthesize a full-length transcript. Promoter escape, which constitutes the initiation-elongation transition, is a key step in regulating productive gene expression. Failure of RNA polymerase to negotiate this transition results in the repetitive synthesis of abortive RNA, a process known as abortive initiation. The extent of abortive initiation vs. promoter escape differs from promoter to promoter and is guided by the initial transcribed sequence (ITS) at each promoter. While previous research has led to the description of a general outline of the promoter escape process, current research will be directed at achieving a detailed mechanism of promoter escape. This goal will require investigation into the sequence-specific nature of promoter escape to understand how an ITS directs a unique abortive initiation-promoter escape program at individual promoters, and controls productive expression by governing the rate of escape and the extent of RNA polymerase partitioning. The proof of a promoter escape mechanism will also require structural characterization of initial transcribing complexes poised on the brink of escape. The methods to be employed in this research will include quantitative in vitro transcription analysis of wild type and mutant promoter templates, kinetic determination of rate and equilibrium constants to understand better the partitioning of transcribing complexes into productive or unproductive conformation, and footprinting techniques to characterize the static or dynamic structure of transcribing complexes. The combined approaches will shed light on how the ITS sequence directs RNA polymerase to relinquish its promoter contacts at specific positions and bring about the escape transition at a defined rate. All of the knowledge gained from studying E. coli RNA polymerase will be of relevance to understanding the promoter escape strategies by T7 RNA polymerase and eukaryotic Pol II.
Broader Impacts: Being affiliated with a women's college, the principal investigator routinely works with many undergraduate students in the research and teaching laboratories. The research provides many self-contained, well-designed, and cutting-edge projects for rigorous and critical investigation. A broad impact of the research projects leads to the training of undergraduates, especially young women and of diverse ethnicity, preparing them for research participation in a scientific career.
Transcription, being the first step of the gene expression process in the cell, is a highly-regulated sequence of events that can be parsed into three phases—initiation, elongation, and termination. Initiation involves the binding of an RNA polymerase enzyme to a promoter—the "ON"—DNA signal to form a catalytic complex that can polymerize NTP substrates into a long RNA in a DNA sequence dependent manner. Regulation exerted at the initiation stage determines how much of a gene product is made, and on many promoters, requires the participation of activator or repressor proteins. Our research has focused on understanding the regulation of transcription initiation at a group of E. coli Eσ70 promoters that are intrinsically strong; that is, their gene expression requires no participation of accessory regulatory proteins, but is dependent on how well the promoter DNA signal agrees with the "maximal" consensus sequence. The better the agreement, the stronger is the binding affinity between RNA polymerase and promoter DNA. This leads to the formation of a highly stable catalytic open complex that initiates RNA synthesis with a high frequency; however, commensurate high level of long RNA synthesis is not obtained. The discrepancy between high initiation frequency and low productive RNA yield was resolved by the observation of abundant abortive RNA synthesis. Investigating this issue on N25, a prototypical "intrinsically strong" promoter, ~95% of all initiation events end in the synthesis and release of abortive RNAs 2-11 nucleotides long; only 5% of initiation result in full-length RNA synthesis. This quantitative analysis showed that the initiation phase on the N25 promoter encompasses transcription to the +11 position where RNAP must undergo the promoter escape maneuver to transition into the elongation phase. If the escape transition is unsuccessful, RNAP abortively initiates—that is, RNAP releases the short abortive transcript, resumes the open complex conformation, and can initiate another round of RNA synthesis again. If the escape transition is successful, RNAP can move away from the promoter region and perform processive elongation downstream. The promoter escape step, therefore, functions as the rate-limiting barrier to the initiation-elongation transition on intrinsically strong promoters. The height of this barrier can be regulated (altered) by changing the promoter recognition region and/or the initial transcribed sequence (ITS) region. Mutations of core promoter elements that deviate from the consensus lowered the stability of the open complex and, consequently, the height of the escape barrier, and vice versa. Changes in the ITS not only delay the position of escape, but also alter the height of the escape barrier. Thus, with N25anti (containing ITS changes from +3 to +20), escape now occurs at the +15/+16 juncture, and the productive yield dwindles to 0.3%. On DG203 (containing ITS changes from +3 to +10), escape does not occur until the +19/+20 juncture, and the productive yield has decreased to 2%. Further, we found that abortive RNAs ≤ 15 nt are susceptible to Gre-factor mediated rescue through cleavage and re-elongation, linking their origin to RNAP backtracking and release. Together, these evidences support the following mechanism of promoter escape: Starting with a highly stable open complex, de novo initiation occurs although polymerase cannot move forward, translocation occurs through scrunching by reeling in denatured DNA to expand the transcription bubble. As initial transcription proceeds, bubble expansion accumulates strain to form highly unstable initial transcribing complexes which can backtrack to produce abortive RNA by rewinding the downstream portion of the expanded bubble. However, at the escape stage, upstream rewinding of the transcription bubble leads to promoter release, forming an elongation complex with a bubble spanning the ITS region. This model is supported by the demonstration of scrunching on the N25 promoter and can explain the role of ITS in changing promoter escape properties. The backtracking-induced abortive mechanism cannot account for the formation of GreB-resistant very long abortive transcripts (VLATs) of 16-19 nucleotides on DG203 promoter. We proposed that GreB-resistant VLATs are the products of RNAP hyper forward translocation during the promoter escape transition and have garnered various supporting evidence for this model through EcoRI-mediated roadblock transcription, Exo III and permanganate footprinting analyses. Further, hyper forward translocation on DG203 is dependent on the very AT-rich spacer DNA/-10 region that contains an embedded UP-like element and participates in GreB-resistant VLAT formation via both αCTD-dependent (involving αCTD-UP-like element contacts) and aCTD-independent (involving intrinsic AT-rich DNA bending/activation) manner. Broader impacts: This grant-funded project provided rich and vigorous training ground for twelve undergraduate- and one postdoctoral researcher, all women. The postdoctoral researcher has pursued a career of science education at the high school level. Of the twelve undergraduate students, six are (will be) pursuing Ph.D. training, four are (will be) pursuing M.D. training, and two are pursuing opportunities in STEM-related fields. Thus, my research effort has contributed to enlarging the human resource infrastructure in the biomedical science.
