In Vitro Properties of RpoS (σ
            <sup>S</sup>
            ) Mutants of
            <i>Escherichia coli</i>
            with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

Gowrishankar, J.; Subbarayan, Pochi R.; Ishihama, Akira

doi:10.1128/jb.185.8.2673-2679.2003

Cited by 22 publications

(18 citation statements)

References 48 publications

(85 reference statements)

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“…2B, Shine-Dalgarno sequence [SD]). Translational reinitiation within the E. coli rpoS gene has also been reported previously (15). To facilitate characterization of the rpoS T2 allele in isogenic backgrounds, the rpoS gene from S. Typhimurium ATCC 14028 was replaced by rpoS T2 , yielding strain 2922KrpoS T2 .…”

Section: Resultsmentioning

confidence: 57%

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

et al. 2010

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Proteins that bind factors typically attenuate the function of the factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor S (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of S -dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a ⌬crl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-S interaction and that residues 1 to 71 in S are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.In bacteria, transcription depends on a multisubunit RNA polymerase (RNAP) consisting of a catalytically active core enzyme (E) with a subunit structure ␣ 2 ␤␤Ј that associates with any one of several factors to form different holoenzyme (E) species. The subunit is required for specific promoter binding, and different factors direct RNAP to different classes of promoters, thereby modulating the gene expression patterns (17). The RNA polymerase holoenzyme containing the 70 subunit is responsible for the transcription of most genes during exponential growth (17). When cells enter stationary phase or are under specific stress conditions (high osmolarity, low pH, or high and low temperatures) during exponential growth, S , which is encoded by the rpoS gene, becomes more abundant, associates with the core enzyme, and directs the transcription of genes essential for the general stress response and for stationary phase survival (17,20,25).Sigma factors compete for binding to a limited amount of the core polymerase (16,17,20,34).70 is abundant throughout the growth cycle and has the highest affinity of all sigma factors for E in vitro (20). In contrast, levels of S reach only about one-third of the 70 levels upon entry into stationary phase, and S exhibits the lowest affinity for E of all sigma factors in vitro (20). The cell uses at least two strategies to ensure the switch between 70 -and S -associated RNA polymerases and to allow gene expression to be reprogrammed upon entry into stationary phase. Several factors (Rsd, 6S RNA, ppGpp, and D...

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Section: Resultsmentioning

confidence: 57%

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

et al. 2010

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“…This would explain why s s activity is retained in suppressor-free strains carrying the amber 33 codon. Indeed, a fragment of s s (rpoSD1-53) lacking the first 53 amino acids is functional in vivo (Subbarayan and Sarkar, 2004) as has previously been shown for a fragment lacking the postulated N-terminal subregion 1.1 (Rajkumari and Gowrishankar, 2002;Gowrishankar et al, 2003). Thus, uncertainties surrounding the ancestral sequence of the rpoS gene, the genetic relationships between different K-12 strains, and the strong selective pressures these strains have encountered in the laboratory, make it difficult to draw conclusions about how common down-mutations (or up-mutations) in rpoS are in the real world outside the laboratory.…”

Section: Resultsmentioning

confidence: 65%

MicroReview: Growth versus maintenance: a trade‐off dictated by RNA polymerase availability and sigma factor competition?

Nyström

2004

Molecular Microbiology

202

189

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SummaryThe regulatory design of higher organisms is proposed to comprise a trade-off between activities devoted to reproduction and those devoted to cellular maintenance and repair. Excessive reproduction will inevitably limit the organism's ability to resist stress whereas excessively devoted stress defence systems may increase lifespan but reduce Darwinian fitness. The trade-off is arguably a consequence of limited resources in any one organism but the nature and identity of such limiting resources are ambiguous. Analysis of global control of gene expression in Escherichia coli suggests that reproduction and maintenance activities are also at odds in bacteria and that this antagonism may be a consequence of a battle between transcription factors for limiting RNA polymerase. The outcome of this battle is regulated and depends on the nutritional status of the environment, the levels of the alarmone ppGpp, and RNA polymerase availability. This paper reviews how the concentration of RNA polymerase available for transcription initiation may vary upon shifts between growth and growth-arrest conditions and how this adjustment may differentially affect genes whose functions relate to reproduction and maintenance.

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“…On the other hand, rpoS from a virulent Salmonella strain (ATCC 14028s) has an ATG start (50). Since it has recently been found that N-terminal deletions of rpoS may retain substantial function (17,36), we tested the function of the S. enterica TTG initiation codon. Site-directed mutations changing the start codon were substituted at the native rpoS locus in the bacterial chromosome as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

Limited Role for the DsrA and RprA Regulatory RNAs in rpoS Regulation in Salmonella enterica

2006

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RpoS, the sigma factor of enteric bacteria that responds to stress and stationary phase, is subject to complex regulation acting at multiple levels, including transcription, translation, and proteolysis. Increased translation of rpoS mRNA during growth at low temperature, after osmotic challenge, or with a constitutively activated Rcs phosphorelay depends on two trans-acting small regulatory RNAs (sRNAs) in Escherichia coli. The DsrA and RprA sRNAs are both highly conserved in Salmonella enterica, as is their target, an inhibitory antisense element within the rpoS untranslated leader. Analysis of dsrA and rprA deletion mutants indicates that while the increased translation of RpoS in response to osmotic challenge is conserved in S. enterica, dependence on these two sRNA regulators is much reduced. Furthermore, low-temperature growth or constitutive RcsC activation had only modest effects on RpoS expression, and these increases were, respectively, independent of dsrA or rprA function. This lack of conservation of sRNA function suggests surprising flexibility in RpoS regulation.RpoS, the general stress and stationary-phase (SP) sigma factor, is highly conserved among Escherichia coli, Salmonella enterica, and other related enteric bacteria. The diverse and often harsh conditions encountered by these bacteria, whether residing as pathogens in the gut or as saprophytes in the environment, require the ability to integrate multiple stress signals and initiate the appropriate cellular responses in order to survive. RpoS serves in this capacity as the master regulator of the general stress response. Its levels increase in response to a number of stress signals, including osmotic shock, nutrient depletion, low temperature, and growth into stationary phase (reviewed in reference 19). As RpoS becomes more abundant, it effectively competes with the vegetative sigma factor in binding to core RNA polymerase, leading to increased transcription of genes necessary for mediating the stress response (49).Regulation of RpoS is complex, with a large posttranscriptional component, and involves trans-acting factors (19). These factors include several small regulatory RNAs (28, 39) which target a cis-acting antisense element within the rpoS mRNA untranslated leader (7). In E. coli, two such small RNAs (sRNAs), DsrA and RprA, activate rpoS translation by binding to and inhibiting the antisense element (reviewed in reference 30). DsrA is necessary for activation of rpoS translation in response to low temperature and osmotic shock (27), while RprA increases RpoS both in response to osmotic shock (29) and in response to a constitutively active rcsC allele, indicating a role in cell envelope stress (15,29).These sRNAs were initially discovered and characterized in E. coli, and their gene sequences are Ϸ90% identical in S. enterica. The high degree of sequence conservation shared by E. coli and S. enterica, in both rpoS and the sRNAs, suggested that their regulatory functions are likely to be conserved as well. Here we describe the results o...

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In Vitro Properties of RpoS (σ ^S ) Mutants of Escherichia coli with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

Cited by 22 publications

References 48 publications

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

MicroReview: Growth versus maintenance: a trade‐off dictated by RNA polymerase availability and sigma factor competition?

Limited Role for the DsrA and RprA Regulatory RNAs in rpoS Regulation in Salmonella enterica

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In Vitro Properties of RpoS (σ S ) Mutants of Escherichia coli with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

Cited by 22 publications

References 48 publications

Identification of Conserved Amino Acid Residues of the Salmonella σ S Chaperone Crl Involved in Crl-σ S Interactions

Identification of Conserved Amino Acid Residues of the Salmonella σ S Chaperone Crl Involved in Crl-σ S Interactions

MicroReview: Growth versus maintenance: a trade‐off dictated by RNA polymerase availability and sigma factor competition?

Limited Role for the DsrA and RprA Regulatory RNAs in rpoS Regulation in Salmonella enterica

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In Vitro Properties of RpoS (σ ^S ) Mutants of Escherichia coli with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions

Identification of Conserved Amino Acid Residues of the Salmonella σ ^S Chaperone Crl Involved in Crl-σ ^S Interactions