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

Abstract

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 following 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 enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and ؊10 region of the promoter on reiterative 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. Usually 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 (typically) 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 additional nucleotide in the transcript, and when transcript slippage occurs repetitively, the same template nucleotide can specify multiple extra residues. This reaction is called reiterative transcription (also known as RNAP stuttering, transcription slippage, and pseudotemplated 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 termination (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 contrast, longer homopolymeric tracts usually are required for reiterative transcription during elongation and termination (6, 7).The physiological significance of reiterative transcription is that it plays a central rol...

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system

“…This indicates that the transcript is leaderless (the start codon is either preceded by only a few nucleotides, as in this case, or it starts directly with a 5-terminal AUG) much like many homologous regulatory C genes ( 27 , 35 , 40 ). The multiple products from the primer extension most likely result from reiterative transcription that may occur during transcription initiation, and can impact gene expression in some cases ( 66 , 67 ). Nevertheless, we found these sequences suboptimal to the consensus sequence: CTAAGA (–35 box) and TATGGC (–10 box).…”

Molecular study on the carAB operon reveals that carB gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri

“…Disruption of the repressors increases the carAB expression levels, which is positively correlated with pyridine production [ 18 ]. A recent work infers that carAB operon in E. coli is regulated through high intracellular levels of UTP that promote reiterative transcription to add extra U residues to the 3′ end of a nascent transcript during transcription initiation [ 19 ].…”

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

“…However, perfect consensus promoters are not actually found in wild-type Escherichia coli cells, and little is known about TSS selection on native promoters and about how variation in promoter sequence affects TSS position. Comparison of the TSS for natural and synthetic Eσ 70 -dependent promoters indicates that a purine 7-8 bp downstream from the last base in the -10 element is typically used for initiation, and in some cases, two or more adjacent positions in an individual promoter are used (1)(2)(3). A pyrimidine at nontemplate strand position −1 is preferred (Fig.…”