Lilian M. Hsu
Promoter escape refers to the latter half of the transcription initiation process. Having formed a highly stable open complex, the RNA polymerase molecule must proceed to relinquish the upstream promoter contacts, release the sigma subunit, translocate downstream, and assume the highly stable and processive elongation conformation, even as the first 10-15 phosphodiester bonds are catalyzed to form an initial transcript. The process of promoter escape is marked with high levels of production of abortive RNAs reflecting the instability of initial transcribing complexes (ITCs). This instability can be viewed as associated with a transitory stage of the enzyme caught between two highly stable conformations, and therefore, subjected to opposing forces--upstream by the promoter elements holding the polymerase back, and downstream by the new contacts that will eventually stabilize the elongation conformation, pulling the polymerase forward. A number of intrinsic and extrinsic factors have been shown to influence the extent of abortive initiation and promoter escape. In this poster, we will address the contribution by the initial transcribed sequence region (ITS; defined as +1 to +20 of the template) as studied with the T5 N25 promoter.
The first evidence that the initial transcribed sequence plays a role in promoter escape was demonstrated by Kammerer et al. (1986) who showed that changing the 18-bp region from +3 to +20 of the T5 N25 promoter to an "anti" sequence (A <--> C and G <--> T, and vice versa) rendered this promoter 10-fold less active in vitro and in vivo without altering the binding constant, Ka. Quantitation of the abortive versus productive yields from these two promoters revealed biochemically that T5 N25antiDSR promoter is 10-fold less effective at promoter escape.
To dissect the contribution of various regions of the ITS, sequence swapping of T5 N25 versus T5 N25antiDSR ITS elements was performed and the promoters transcribed. The outcome of this analysis showed that while the proximal ITS (+1 to +10) played a major role in determining the abortive product pattern, the distal ITS (+11 to +20) also affected the efficiency of promoter escape.
Focusing on the proximal ITS of T5 N25, single nucleotide replacement mutants from +2 to +9 were constructed. Transcriptional analysis of the productive versus abortive yields from these mutant promoters showed that, in general, changing a single nucleotide in the ITS led to only small changes (less than 2 fold) in full-length RNA synthesis, with the exception of several specific mutations. Analysis of the specific mutants T2A, A3T, and G9T revealed a KM-dependence of [NTP] on full-length RNA synthesis. Quantitation of abortive yields of the transcripts immediately affected by the base change suggests that the strength of the last RNA-DNA base pair is a factor influening the stability of the ITC. Detailed analysis of the specific mutations will be presented.