In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 2. Formation and characterization of two distinct classes of initial transcribing complexes.

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.