Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the
            <i>groE</i>
            Promoter

Wang, Yan; deHaseth, Pieter L.

doi:10.1128/jb.185.19.5800-5806.2003

Cited by 28 publications

(40 citation statements)

References 34 publications

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“…Although similar to the functional 32 binding sequence deduced from the E. coli groE promoter (36), this sequence shows a number of differences, particularly and most importantly the identification of position Ϫ7 as relevant for recognition by 32 . Wild-type Pm activity in the rpoS mutant background was 60% of its value in MC4100 (7,000 MU) in the stationary phase.…”

Section: At Pm As 5ј-(h)ccc(s)(k)ta(d)g(m)t-3ј (Positions ϫ18mentioning

confidence: 76%

RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

Domı́nguez-Cuevas¹,

Marín

Ramos

et al. 2005

Journal of Biological Chemistry

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The Pm promoter of the benzoate meta-cleavage pathway is transcribed with E 32 or E 38 according to the growth phase, with an identical transcriptional start site. To investigate sequence determinants in the interaction between either of the two RNA polymerases and Pm, all possible single mutants between positions ؊7 and ؊18 were generated, and the activity in the exponential and stationary phases of the resulting mutant promoters was compared. The results precisely delimited a ؊10 element between positions ؊7 and ؊12 (TAGGCT), which defined a promoter sharing nucleotides with both 38 and 32 consensus. The first two and the last positions of this hexamer were crucial for recognition by both polymerases. Position ؊10 was the only one specifically recognized by E 38 , whereas positions ؊8, ؊9, and the C-track between positions ؊14 and ؊17 were important for specific E 32 recognition. Western blots showed that 32 was only detectable in the exponential phase, and 38 appeared in the early stationary phase. In the rpoH mutant KY1429, 38 was already present in the exponential growth phase both free and bound to the RNA polymerase core, in good correlation with the transcription levels found. Pm seems to be optimized for recognition by 32 as an initial response to the addition of effector to the medium and allows binding of the adaptable 38 -dependent RNA polymerase in the stationary phase. XylS is always required for Pm transcription. Therefore, the mechanism that controls Pm expression involves specific nucleotide sequences, the abundance of free and core-bound 32 and 38 factors during growth, and the presence of the regulator activated by an effector.

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Section: At Pm As 5ј-(h)ccc(s)(k)ta(d)g(m)t-3ј (Positions ϫ18mentioning

confidence: 76%

RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

Domı́nguez-Cuevas¹,

Marín

Ramos

et al. 2005

Journal of Biological Chemistry

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“…The A264R substitution reduced the activity of 32 with the wt promoter to only 70% that of the wt 32 , despite the fact that the substituted arginine side chain was much larger than the alanine it replaced. The A-32C promoter substitution had previously been shown to reduce the activity seen with wt 32 to 65% of that obtained with the wt promoter (35). However, when combined, the A264R substitution in 32 and the A-32C substitution in the promoter resulted in an activity of 83% of that seen with wt 32 and the promoter (Fig.…”

Section: Fig 3 Effect Of Single Amino Acid Mutation Onmentioning

confidence: 79%

“…To assess the importance of particular amino acid residues for 32 function, a collection of variants was constructed and expressed from pSAKT32, which results in approximately physiological intracellular levels of 32 protein upon IPTG induction (35). The selection of amino acids for substitution was based on a comparison of the 70 and 32 sequences (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition

2005

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Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor 32 rise greatly, resulting in the increased formation of the 32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on 32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving 32 expression from one plasmid and a ␤-galactosidase reporter gene driven by the 32 -dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on 32 activity. It was found that the recognition of the ؊35 region involves similar amino acid residues in regions 4.2 of E. coli 32 and 70 . Three conserved amino acids in region 2.3 of 32 were found to be only marginally important in determining activity in vivo. Differences between 32 and 70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.The master regulator for the heat shock response in Escherichia coli, now usually referred to as 32 , was identified as a sigma factor over 20 years ago (11,18). When E. coli cells are exposed to high temperatures (e.g., 42°C), the levels of 32 first rise steeply by a variety of different mechanisms and then level off at about twice the initial level (6,10,25). 32 binds to core RNA polymerase (RNAP) and directs the RNAP to the heat shock promoters, for which the consensus sequence differs from those utilized by RNAP containing the housekeeping sigma factor, 70 (4, 10, 35, 36, 38). The difference is pronounced in the Ϫ10 region, but in the Ϫ35 region the two classes of promoters have a 4-base-pair sequence in common. In view of the homology between the two proteins in region 4.2 (see Fig. 1A), it had been postulated early on that the recognition of the Ϫ35 regions would involve similar amino acids of 32 and 70 (30). While the most evident role of 32 is in directing the expression of genes allowing the cell to deal with the consequences of heat shock, it is an essential protein at physiological (37°C) temperatures (37). Despite its importance to the survival of the cell, few studies have addressed the roles of particular amino acids. Amino acids in region 2.1 were found to markedly affect the stability of 32 in vivo (12); a stretch of amino acids between regions 2 and 3 of 32 is known to participate in the binding of DnaK (21), which functions as an anti-sigma factor of 32 ; and amino acids that play a role in the interaction of 32 with the core have been identified (13,14). In addition, random linker insertion mutagenesis has provided information concerning the roles of various regions of 32 in determining its activity (21). Here we report the results of targeted mutagenesis of amino acids in variou...

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“…In Escherichia coli, variants with 14-bp spacers display the highest level of transcriptional induction upon heat shock, $2.5-fold higher than promoters with 13-bp spacers (Wang and Dehaseth 2003). Since the consensus promoter and the Sigma factor binding sites are identical for E. coli and Buchnera (Wilcox et al 2003), the longer spacer length may increase ibpA expression and thermal tolerance.…”

Section: Resultsmentioning

confidence: 99%

Dynamics of a Recurrent Buchnera Mutation That Affects Thermal Tolerance of Pea Aphid Hosts

et al. 2010

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Mutations in maternally transmitted symbionts can affect host fitness. In this study we investigate a mutation in an obligate bacterial symbiont (Buchnera), which has dramatic effects on the heat tolerance of pea aphid hosts (Acyrthosiphon pisum). The heat-sensitive allele arises through a single base deletion in a homopolymer within the promoter of ibpA, which encodes a universal small heat-shock protein. In laboratory cultures reared under cool conditions (20°), the rate of fixation (1.4 3 10 À3 substitutions per Buchnera replication) is much higher than the previously estimated mutation rate for single base deletions in homopolymers in the Buchnera genome, implying a strong selective benefit. This mutation recurs in natural populations, but seldom reaches high frequencies, implying that it is only rarely favored by selection. Another potential source of physiological stress in pea aphids is infection by other microorganisms, including facultative bacterial symbionts, which occur in a majority of pea aphids in field populations. Frequency of the heat-sensitive Buchnera allele is negatively correlated with presence of facultative symbionts in both laboratory colonies and field populations, suggesting that these infections impose stress that is ameliorated by ibpA expression. This single base polymorphism in Buchnera has the potential to allow aphid populations to adapt quickly to prevailing conditions.

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Sigma 32-Dependent Promoter Activity In Vivo: Sequence Determinants of the groE Promoter

Cited by 28 publications

References 34 publications

RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

RNA Polymerase Holoenzymes Can Share a Single Transcription Start Site for the Pm Promoter

Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition

Dynamics of a Recurrent Buchnera Mutation That Affects Thermal Tolerance of Pea Aphid Hosts

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