In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 1. RNA Chain Initiation, Abortive Initiation, and Promoter Escape at Three Bacteriophage Promoters

Abstract

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.

Internal Initiation of Influenza Virus Replication of Viral RNA and Complementary RNA in Vitro

“…In particular, 50 M UTP limited the formation of the stable initiation complex from the 3Ј-end of c84 (Figs. 6 and 7), which was similar to the abortive initiation of T7 and Escherichia coli RNA polymerases (37,38). This UTP sensitivity is consistent with high primer activity of AGU in the case of c84 (Fig.…”

Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

“…S1). We note the accumulation of abortive RNA products in reactions performed with template #2, which we attribute to the two base pair substitutions in the initial transcribed region; initial transcribed region alterations can have dramatic effects on abortive transcript production (Hsu et al 2003;Chander et al 2007). The ;116-nt terminated transcript (T) is indicated, and the asterisk (*) indicates a shorter terminated transcript that is the result of transcription initiating under the control of the promoter-distal extended À10 element.…”

The transition from transcriptional initiation to elongation

“…In E. coli, such differences can affect both the affinity of RNAP for the promoter and the rate of post-recruitment steps [37], such as the level of abortive initiation and promoter escape [38,39], the formation of unproductive "moribund" RNAP complexes [40,41], and σ-dependent pausing of RNAP in the initial transcribed region [42,43]. As initial transcription occurs through scrunching, an intriguing possibility is that the presence of promoter-proximal σ-dependent pause sites may cause RNAP to favor forward translocation and hence promoter escape, rather than repeated cycles of abortive initiation.…”