Abortive initiation refers to the process associated with the early stage of the transcriptional cycle when an engaged RNA polymerase molecule attempts to transit from the initiation phase to the elongation phase. When this transition is unsuccessful, the oligonucleotide product (2-9 nucleotides long) is aborted and the RNA polymerase cycles back to the transcription initiation point to start over. The ease with which this transition occurs defines the rate of promoter clearance by the enzyme molecule; different E. coli promoters apparently undergo repetitive cycling to different degrees. Recently, the promoter-clearance step has been shown to be a target of gene regulation. The broad goal of this study is aimed at achieving a unifying and sequence-specific understanding of abortive initiation. Such an understanding is crucial for a full description of the RNA polymerase catalytic mechanism and differential gene expression from individual promoters. Although this study will be conducted with the E. coli polymerase-promoter systems, the findings will be relevant to understanding gene expression in all organisms. Specifically, four experimental approaches will be employed in this study. One, a large set of frequently-studied E. coli promoters will be surveyed by quantitative and qualitative in vitro transcriptional assay to determine the extent and the pattern of abortive initiation under similar reaction conditions. Such an extensive survey may reveal sequence-specific features in the initial transcribed sequence (ITS) region important for abortive initiation. Two, the in vivo activities of the aforementioned promoter set can be determined from fusion constructs by measuring the reporter enzyme chloramphenicol acetyl transferase (CAT) activity as well as by quantitating the steady-state level of CAT mRNA by nuclease-protection assays. The above two lines of investigation will lead to an in vivo-in vitro promoter strength correlation which now takes into account the kinetics of promoter clearance. Three, by cassette mutagenesis and sequence-swap experiments between a highly-abortive vs. a poorly-abortive promoters, the sequence region(s) in an """"""""extended"""""""" promoter [core promoter + UAS (upstream activating sequence) + ITS] important for abortive initiation can be pinpointed. Ibis type of assignment forms the basis for further assessment of the contribution of individual nucleotides to abortive initiation. Four, to deduce the molecular differences responsible for abortive vs. productive initiation, footprinting analyses will be performed to compare the polymerase-promoter interactions in highly-abortive vs. poorly-abortive promoters. Together, these analyses should forge a clear understanding of the mechanism of abortive initiation.
| Hsu, Lilian M; Vo, Nam V; Kane, Caroline M et al. (2003) In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters. Biochemistry 42:3777-86 |
| Hsu, L M (1996) Quantitative parameters for promoter clearance. Methods Enzymol 273:59-71 |
