Localization of the intrinsically bent DNA region upstream of the<i>E.coli rrnB</i>P1 promoter

Gaál, Tamás; Lin, Rongcheng; Estrem, Shawn T.; Yang, Jin; Wartell, Roger M.; Gourse, Richard L.

doi:10.1093/nar/22.12.2344

Cited by 43 publications

(29 citation statements)

References 38 publications

(52 reference statements)

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“…Mutants with an altered position Ϫ37 were removed from the analysis due to a 10-fold inhibitory effect (data not shown). Presumably, modification of this position affects core promoter recognition (7,10).…”

Section: Resultsmentioning

confidence: 99%

The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription

1997

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The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from ؊38 to ؊50 [the ؊45 region]). The possible roles of the ؊45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the ؊40 to ؊45 sequence and required an intact RNA polymerase ␣ subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The ؊40 to ؊45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.Transcription is a multistep process that begins with the binding of RNA polymerase (RNAP) to specific DNA sites termed promoters. A majority of Escherichia coli promoters (those recognized by the 70 holoenzyme) exhibit a general structural pattern consisting of two conserved hexameric sequences separated by approximately 17 bp, termed the core promoter. The strength of RNAP-core promoter binding, often correlated with the level of transcription, is determined by a number of factors. The major factors include the match of the promoter hexamers and DNA spacer to the established consensus sequence (9), the presence of activator binding sites (1) and functional activator proteins, and the presence of an upstream DNA recognition element (called an UP element) in some strong promoters, such as rrnB P1 and rrnB P2 (25; for a review, see reference 23). The UP element is recognized by the carboxyl-terminal domain of the RNAP ␣ subunit (␣-CTD).E. coli lac P1 is a well-studied 70 promoter containing a relatively weak core promoter. In addition, the sequence immediately upstream of the core promoter (Ϫ38 to Ϫ50, hereinafter called the Ϫ45 region) is a poor match to the sequences of UP elements frequently associated with highly active promoters (25). lac P1 is highly regulated, being subject to both repression and activation. Repression is maintained by a functional lacI gene product, coincident with the absence of a suitable inducer. Activation is achieved...

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Section: Resultsmentioning

confidence: 99%

The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription

1997

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“…13 amino acids (ϳ46 Å if fully extended) (25), ␣CTD should not be able to reach further upstream than positions ϳϪ80 to Ϫ90 in the absence of DNA distortion (36,38). Assuming FIS bound at the distal sites exerts its effects on transcription by interacting directly with RNAP, we suggest that FIS bound at the proximal site(s), possibly in conjunction with intrinsic DNA curvature (20), distorts the DNA to facilitate these contacts. Differences in the positions of the upstream FIS sites at different operons, differences in the angles of the bends induced by FIS bound to its various binding sites, and differences in intrinsic DNA curvature in different operons could all contribute to differences in the extents of activation by the different rrn P1 upstream regions.…”

Section: Discussionmentioning

confidence: 95%

Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven rrn P1 Promoters in Escherichia coli

et al. 2001

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The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.The synthesis of ribosomes in bacteria is determined by the rate of synthesis of rRNA and, at high growth rates in Escherichia coli, rRNA promoters account for more than half of the transcription in the cell (10). The large contribution of rRNA transcription to total cellular transcription, the central role played by ribosomes in cell physiology, and the importance of rRNA regulation as a model for the control of global gene expression justify intensive analysis of rRNA promoters.rRNA is transcribed from two promoters, P1 and P2, at each of the seven rrn operons: rrnA, rrnB, rrnC, rrnD, rrnE, rrnG, and rrnH. However, most previous work on the factors that contribute to the unique strength and regulation of the rrn P1 promoters has been limited to rrnB P1. The rrnB P1 core promoter contains near-consensus Ϫ35 and Ϫ10 hexamers. Interactions between RNA polymerase (RNAP) and the rrnB P1 core promoter (defined here as Ϫ41 to ϩ1 with respect to the transcription start site) are regulated by the concentration of the initiating nucleotide (19) and by the concentration of guanosine tetraphosphate (ppGpp), a nucleotide that inhibits rRNA synthesis in response to amino acid starvation (5, 12). However, the core promoter accounts for Ͻ1% of the activity of the full-length promoter (defined here as containing rrnB P1 sequence upstream to Ϫ154) (21, 22, 41). The strength of the full-length promoter is attributable to two features: (i) the UP element, an AϩT-rich region of DNA located upstream of the Ϫ35 hexamer and recognized by the carboxy-terminal domain of the ␣ subunit (␣CTD) of RNAP (44); and (ii) FIS, an 11.2-kDa DNA-binding protein that binds as a dimer upstream of the UP element (46, 54), bends each of its binding sites 40 to 90°(18), and interacts with the ␣CTD of RNAP to activate transcription (8).UP elements are found at both rRNA (31,44,47,54) and non-rRNA (23) promoters, an...

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“…We also tested growth rate regulation of a class of rrnB P1 mutants with reduced promoter activities, using system II promoter-lacZ fusions. A substitution just upstream of the -35 hexamer [C-37T] severely reduced promoter activity without affecting upstream activation (22), possibly by creating a T tract that alters the DNA structure of this region (13). The [T-14A] and [G-34T] mutations also severely reduced promoter activity (11), most likely by interfering with interactions with the sigma subunit of RNA polymerase, since the mutations are at highly conserved positions in the -10 and -35 hexamers, respectively.…”

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confidence: 99%

Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli

Bartlett

Gourse

1994

J Bacteriol

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We have extended our previous studies of the DNA sequences required for growth rate-dependent control of rRNA transcription in Escherichia coli. Utilizing a reporter system suitable for evaluation of promoters with low activities, we have found that the core promoter region of mnB P1 (-41 to + 1 with respect to the transcription initiation site) is sufficient for growth rate-dependent control of transcription, both in the presence and in the absence of guanosine 3'-diphosphate 5'-diphosphate (ppGpp). The core promoter contains the -10 and -35 hexamers for recognition by the sigma 70 subunit of RNA polymerase but lacks the upstream (UP) element, which increases transcription by interacting with the alpha subunit of RNA polymerase. It also lacks the binding sites for the positive transcription factor FIS. Thus, the UP element, FIS, and ppGpp are not needed for growth rate-dependent regulation of rRNA transcription. In addition, we find that several core promoter mutations, including -10 and -35 hexamer substitutions, severely reduce rrnB P1 activity without affecting growth rate-dependent control. Thus, a high activity is not a determinant of growth rate regulation of rRNA transcription.In steady-state cultures of Escherichia coli, the synthesis of rRNA per total cell protein increases with the square of the growth rate of the cell, a relationship called growth ratedependent control (24). Growth rate control acts at the level of transcription initiation from the P1 promoters of the seven rRNA (rrn) operons, while the downstream P2 promoters of these operons appear to be transcribed constitutively (15, 32). A feedback system senses the level of translationally competent ribosomes and controls transcription initiation at rrn P1 promoters and some tRNA promoters in response to the need for protein synthesis (7,17,21,27,37). It has been suggested that this feedback system is the mechanism by which growth rate control of transcription is achieved (15, 27), although alternative hypotheses have been proposed (2,3,(18)(19)(20), and the topic remains controversial.Another system, stringent control, also acts at the level of transcription initiation from the rm P1 promoters (16,23,33). In this system, rRNA synthesis is rapidly and severely inhibited by the production of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) in response to aminoacyl-tRNA limitation (6, 34), although the mechanism by which ppGpp acts to reduce transcription is still unclear.Less is understood about the trans-acting factors involved in growth rate regulation of rm P1 transcription. Since ppGpp concentrations are inversely proportional to the growth rate of the cell under most conditions, ppGpp was proposed to be responsible for growth rate-dependent control (6, 31). However, it was found that rRNA transcription increases normally with growth rate even in strains lacking ppGpp (12) and that transcription from rRNA promoters can vary without corresponding changes in ppGpp levels (8). The FIS protein, another trans-acting factor that affects rn P1 trans...

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Localization of the intrinsically bent DNA region upstream of theE.coli rrnBP1 promoter

Cited by 43 publications

References 38 publications

The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription

The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription

Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven rrn P1 Promoters in Escherichia coli

Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli

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