UTP Allosterically Regulates Transcription by Escherichia coli RNA Polymerase from the Bacteriophage T7 A1 Promoter Ronald S. Johnson* and Rebecca E. Chester Department of Biochemistry Brody School of Medicine at East Carolina University Greenville, NC 27858-4354 USA In the case of Escherichia coli RNA polymerase, UTP at elevated concen- trations suppresses terminated transcript accumulation during multiple- round transcription from a DNA construct containing the T7 A1 promoter and T e terminator. The step that is affected by UTP at elevated concen- trations is promoter clearance. In an attempt to understand better the mechanism by which UTP regulates this step, we analyzed the effect of UTP on the formation of pppApU in the presence of only UTP and ATP. At elevated concentrations, UTP is a non-competitive inhibitor with respect to ATP in the formation of pppApU. This indicates that the effect of UTP on the formation of pppApU is mediated through an allosteric site. Moreover, the magnitude of the inhibition of pppApU formation is sufficient to account for the decrease in terminated transcript accumu- lation at elevated UTP concentrations. Thus, it appears that UTP modulates terminated transcript accumulation during multiple-round transcription from this DNA construct by allosteric regulation of promoter clearance at the point of transcription initiation. q 2002 Elsevier Science Ltd. All rights reserved Keywords: Escherichia coli RNA polymerase; promoter clearance; transcription initiation; allosteric regulation; transcriptional pausing *Corresponding author Introduction RNA synthesis as catalyzed by Escherichia coli RNA polymerase is a complex multistep process. The first step is promoter search and open complex formation. This is followed by promoter clearance which involves all of the steps up to and including the formation of a stable ternary elongation com- plex consisting of the enzyme, the DNA template and the nascent RNA transcript. The process of elongation continues until a termination site is reached. At this point, the full-length RNA tran- script is released along with the enzyme. Regu- lation may occur at any step along this pathway and may involve accessory proteins. 1–7 In most cases, the role of nucleoside tri- phosphates in regulating the synthesis of full- length transcripts has been postulated to be mediated in a passive way by variations in their affinities for the active site at different points along the template. 4,8 – 12 To gain further insight into the role of nucleoside triphosphates in regu- lating the synthesis of full-length transcripts, we used several DNA constructs containing either just the T7 A1 promoter or the T7 A1 promoter along with the T7 T e terminator. We investigated the effects of ATP and UTP on the production of full-length transcripts. These two nucleotides are involved in transcription initiation at the T7 A1 promoter. At elevated concentrations, UTP but not ATP suppressed the formation of full-length tran- scripts during multiple-round transcription from the DNA construct that contained the A1 promoter and the T e terminator. The data obtained here are consistent with a model in which UTP regulates transcription through an allosteric site. Results The accumulation of transcripts from the A1 promoter is modulated by UTP levels during multiple-round transcription The RsaI– SalI fragment from plasmid pARl707 contains the A1 promoter along with the T e termi- nator from bacteriophage T7. The 5 0 terminal 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: johnsonro@mail.ecu.edu Abbreviations used: CIP, calf intestinal alkaline phosphatase. doi: 10.1016/S0022-2836(02)00042-6 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 318, 305–320