Transcription Start Site Sequence and Spacing between the 10 Region and the Start Site Affect Reiterative Transcription-Mediated Regulation of Gene Expression in Escherichia coli Xiaosi Han,* Charles L. Turnbough, Jr. Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3= end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3= end of a nascent transcript during transcription initiation. Immediately follow- ing the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first en- zyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucle- otide sequence at and near the transcription start site and spacing between the start site and 10 region of the promoter on reit- erative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation. U sually during transcription, the nascent RNA transcript and the template strand of DNA move in tandem as an RNA- DNA hybrid. However, during transcription of a homopolymeric tract in the DNA template, the nascent transcript can slip (typi- cally) one base upstream without movement of the DNA template within the active site of RNA polymerase (RNAP) (1, 2). This repositioning allows the same template base to specify an addi- tional nucleotide in the transcript, and when transcript slippage occurs repetitively, the same template nucleotide can specify mul- tiple extra residues. This reaction is called reiterative transcription (also known as RNAP stuttering, transcription slippage, and pseu- dotemplated transcription) and appears to be catalyzed by all RNAPs (2–5). Reiterative transcription can involve the repetitive addition of any of the four nucleoside triphosphate substrates and occurs during transcription initiation, elongation, and termina- tion (2, 3, 6, 7). During initiation, when the length of the RNA- DNA hybrid can be shorter than that of the 8- to 9-bp hybrid that forms during elongation (8), a homopolymeric tract as short as three residues can enable reiterative transcription (9, 10). In con- trast, longer homopolymeric tracts usually are required for reiter- ative transcription during elongation and termination (6, 7). The physiological significance of reiterative transcription is that it plays a central role in regulating the expression of numerous prokaryotic, viral, and eukaryotic genes through an assortment of different mechanisms (3, 11). In Escherichia coli, expression of at least five operons is regulated by mechanisms involving the reit- erative addition of U residues during transcription initiation (4, 12–15). Although these mechanisms can differ fundamentally (11), three examples include analogous mechanisms that regulate, in part, the expression of the pyrBI and carAB pyrimidine nucle- otide biosynthetic operons and the galETKM (gal) galactose cata- bolic operon (12–14). The promoters of each of these operons (at least one when there are multiple promoters) include initially transcribed regions that contain a tract of three T·A base pairs located one or two bases downstream from the transcription start site (Fig. 1). These homopolymeric tracts, here referred to as (non- template strand) T tracts, enable a fraction of the nascent tran- scripts to enter the reiterative pathway. Entry into this pathway is enhanced by high intracellular concentrations of UTP (i.e., the reiterative substrate), resulting in a larger fraction of transcripts containing extra U residues. In each case, the addition of extra U residues causes the release of the nascent transcript from the tran- scription initiation complex, thereby repressing operon expres- sion. This repression allows the cell to use UTP levels to control pyrBI, carAB, and gal expression and synthesize the encoded en- zymes at optimal levels for growth. Although these control mech- anisms are basically the same, the range of regulation afforded by them varies considerably, specifically, 2-fold, 3-fold, and 7-fold for the gal, carAB, and pyrBI operons, respectively (12–14). These different ranges presumably reflect important differences in pro- moter sequences, which could include different sequences at or near the transcription start site or the spacing between the start site and the 10 region (Fig. 1). In this study, we constructed mutant variants of the carAB P1 promoter, one of two carAB promoters and the only one that participates in reiterative transcription-mediated gene regulation Received 9 April 2014 Accepted 27 May 2014 Published ahead of print 2 June 2014 Address correspondence to Charles L. Turnbough, Jr., chuckt@uab.edu. * Present address: Xiaosi Han, Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama, USA. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.01753-14 2912 jb.asm.org Journal of Bacteriology p. 2912–2920 August 2014 Volume 196 Number 16