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.
Hsu
Lilian M
LM
Program in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts 0l075-6456, USA. lhsu@mtholyoke.edu
Vo
Nam V
NV
Kane
Caroline M
CM
Chamberlin
Michael J
MJ
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012318:RNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013604:T-Phages / Q000235:genetics; D024363:Transcription Initiation Site / Q000502:physiology*; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D012329:RNA, Bacterial; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012318:RNA Polymerase I; D014544:Uridine Triphosphate