Sequences upstream of the −35 hexamer of rrnB P1 affect promoter strength and upstream activation

Josaitis, Cathleen A.; Gaál, Tamás; Ross, Wilma; Gourse, Richard L.

doi:10.1016/0167-4781(90)90186-6

Cited by 45 publications

(35 citation statements)

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“…Accordingly, the UASs of the tyrT, metY, thrU (tufB), and rrnB operons all bind one and the same protein (25,33). Sequences upstream of the P1 promoters of all rRNA operons and the promoters of 13 tRNA operons (but not all tRNA operons) match the consensus sequence for FIS-binding sites (18,33). Recently Ross et al (18,27) independently demonstrated that FIS acts as the trans activator of the rrnB operon.…”

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confidence: 99%

“…Sequences upstream of the P1 promoters of all rRNA operons and the promoters of 13 tRNA operons (but not all tRNA operons) match the consensus sequence for FIS-binding sites (18,33). Recently Ross et al (18,27) independently demonstrated that FIS acts as the trans activator of the rrnB operon. In this context the question of under which conditions the trans activation control system becomes operative arises.…”

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FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions

et al. 1992

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In Escherichia coli transcription of the tRNA operon thrU (tuJB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tuJB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tuiB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed.Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tuJB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that of wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is independent of nutritional factors. The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation. FIS allows very fast bacterial growth.The synthesis of rRNA of Escherichia coli is finely tuned to the cell's environmental conditions. Cells growing in a constant environment do not show a significant turnover or a significant buildup of free rRNA or vacant ribosomes, except at very low growth rates (for reviews, see references 20, 21, and 26). Consequently, ribosomes are utilized at maximal or near-maximal capacity. Upon alteration of the nutritional capacity of the medium, leading to a different but constant environment, cells promptly readjust the synthesis of their rRNA and tRNA to meet the demands of an altered growth rate. In exponentially growing cells the concentration of ribosomes (and of rRNA) thus appears to be proportional to growth rate (6,8...

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FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions

et al. 1992

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“…Therefore, if the A/T tracts are responsible for the small degree of curvature associated with this region, either the mutations do not affect this curvature, or the curvature plays little role in the 30-fold increase in transcription resulting from UP element function. On the other hand, either displacement of the UP element by non-integral portions of a helical turn (6,29,34) or creation of an additional T tract with a T substitution at position -37 (29,34) severely decreases promoter activity. Thus, correct positioning of the RNAP alpha subunit (which binds to the UP element) relative to the sigma subunit (which binds to the -10 and -35 hexamers) most likely plays a major role in promoter activity (2).…”

Section: Discussionmentioning

confidence: 99%

“…The A/T-tract mutations reduce transcription only 1.3 to 1.6 fold, implying that the continuous runs of A or T residues do not account for the 30-fold effect of the UP element. Altered DNA structure from a mutation at - 37 We have reported previously that a mutation at position -37 (C-37T) reduces rrnB P1 promoter activity 10-20 fold, but that substitution of an A or G residue at this position reduces activity only 2-3 fold (29,34). We speculated that the large allele-specific effect of the T substitution might result not from the importance of the wild type C residue to promoter function, but from the creation of a series of four T residues in phase with another T tract upstream, which might result in a bend that might be detrimental to promoter function in vivo.…”

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

Gaál¹,

Lin²,

Estrem³

et al. 1994

Nucl Acids Res

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DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold In vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

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E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

et al. 1990

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An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20‐ to 30‐fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10‐ to 20‐fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1‐lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5‐fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5‐fold in a Fis‐ strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

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Sequences upstream of the −35 hexamer of rrnB P1 affect promoter strength and upstream activation

Cited by 45 publications

References 23 publications

FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions

FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions

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

E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

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