Two Extracytoplasmic Function Sigma Subunits, ς
 E
 and ς
 FecI
 , of
 Escherichia coli
 : Promoter Selectivity and Intracellular Levels

Maeda, Hiroto; Jishage, Miki; Nomura, Tasuku; Fujita, Nobuyuki; Ishihama, Akira

doi:10.1128/jb.182.4.1181-1184.2000

Cited by 51 publications

(39 citation statements)

References 36 publications

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“…The protein composition of the E. coli nucleoid changes markedly depending on its cell growth phase . Likewise, the molecular composition of the transcription apparatus (Jishage & Ishihama 1995;Jishage et al 1996;Maeda et al 2000), and translation machinery (Wada et al 1990;Yamagishi et al 1993) changes depending on the cell growth phase. Thus, we extended our analysis so as to observe the growth phase-coupled change in the intracellular localization of the test proteins.…”

Section: Indirect Immuno¯uorescent Observation Of Dna-binding Proteinsmentioning

confidence: 99%

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

2000

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Background: The genome DNA of Escherichia coli is folded into the nucleosome-like structure, often called a nucleoid, by the binding of several DNAbinding proteins. We previously determined the speci®city and af®nity of DNA-binding for 12 species of the E. coli DNA-binding protein, and their intracellular concentrations at various growth phases. The intracellular localization of these proteins in E. coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA-binding proteins.

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Section: Indirect Immuno¯uorescent Observation Of Dna-binding Proteinsmentioning

confidence: 99%

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

2000

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“…Estimation of RNAP partitioning between promoters, nonspecific binding sites on DNA, and the cytoplasm requires knowledge of the levels of E, 70 (the housekeeping factor), and at least some alternative s. However, published values have been measured by different techniques, in different strains, and under different physiological conditions (both during exponential phase and during entry into stationary phase) (18)(19)(20)(21)(22)(23)(24)(25)(26). In fact, discrepancies in these numbers have led to the common perceptions that (i) as most RNAP is elongating, only a minor fraction could be bound to DNA nonspecifically (27,28), and (ii) cellular levels of 70 are much less than that of RNAP (29); thus, s may compete because of their excess over free RNAP.…”

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confidence: 99%

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

Grigorova¹,

Phleger

Mutalik

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

163

155

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To explore scenarios that permit transcription regulation by activator recruitment of RNA polymerase and competition in vivo, we used an equilibrium model of RNA polymerase binding to DNA constrained by the values of total RNA polymerase (E) and 70 per cell measured in this work. Our numbers of E and 70 per cell, which are consistent with most of the primary data in the literature, suggest that in vivo (i) only a minor fraction of RNA polymerase (<20%) is involved in elongation and (ii) 70 is in excess of total E. Modeling the partitioning of RNA polymerase between promoters, nonspecific DNA binding sites, and the cytoplasm suggested that even weak promoters will be saturated with E 70 in vivo unless nonspecific DNA binding by E 70 is rather significant. In addition, the model predicted that s compete for binding to E only when their total number exceeds the total amount of RNA polymerase (excluding that involved in elongation) and that weak promoters will be preferentially subjected to competition.nonspecific DNA binding ͉ gene regulation promoter ͉ systems biology ͉ Escherichia coli I n bacteria transcription is initiated by RNA polymerase (RNAP) holoenzyme (E ), which is formed when core RNAP (E) binds the transcription initiation factor (1). E initially binds to promoter sites in a closed complex, which then transits to an open complex, competent for transcription. The number of intermediates between the closed and open complex is variable and promoter-dependent; each step may be subject to regulation in vivo (2, 3). At least for some promoters, E binding to promoters is thought to be reversible on the time scale of transcription initiation in vivo (3); reversibility has also been demonstrated in vitro for several promoters (3-6). Even binding to the strong lac UV5 promoter is reversible in vitro when tested under conditions that approximate the in vivo situation (6).Recruitment of E to promoters in vivo is thought to depend on the intrinsic binding affinity of the promoter and is modulated by repressors that prevent and activators that stabilize interactions between E and the promoter (3). Based on in vitro studies of the mechanism of activator function, it is believed that promoters that bind E weakly require activators to recruit E . In addition, cells contain multiple s, which direct E to various sets of promoters specific to the factors (1). These s are believed to compete with each other for binding to E (7-10). By changing the relative levels of the s, Escherichia coli is thought to coordinate its transcriptional program with growth conditions (11-13). This view is based on observations indicating that (i) overexpressing one decreases expression of genes controlled by another (7), (ii) mutationally altering binding constants of one for E alters expression by another (14), and (iii) physiological effectors such as ppGpp may act by altering relative binding of s to E (8-10). In the present work, we use an equilibrium model of RNAP binding to DNA to explore in vivo scenarios that permit transcription reg...

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“…The intracellular concentrations of all seven sigma factors in growing cells of E. coli W3110 have been determined (Jishage & Ishihama, 1995;Jishage et al, 1996;Maeda et al, 2000). Since then we have been involved in determination of the intracellular concentrations of the 300 TFs using two approaches: (1) quantitative immunoblot analysis and (2) reporter assay of promoters of the genes encoding TFs.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, for the accurate estimation of intracellular levels of the regulatory proteins for transcription, all the test proteins must be measured using the same cultures of the same bacterial strains and the same experimental systems. We then systematically measured intracellular concentrations of sigma subunits and TFs using two approaches: quantitative immunoblot analyses using anti-sigma (Jishage & Ishihama, 1995;Jishage et al, 1996;Maeda et al, 2000) or anti-TF antibodies (Ishihama, et al, 2014); and quantification of promoter activity using the promoter assay vector of the fluorescent protein reporter system (Shimada et al, 2005). In this paper, we describe the determination of intracellular levels of 90 TFs of six TF families by using the TF promoter-GFP translation fusion vectors.…”

Section: Introductionmentioning

confidence: 99%

“…In E. coli K-12, seven species of the sigma subunit exist, each recognizing a specific set of promoters. Some of the recognition target genes have been characterized for each sigma subunit using standard molecular genetics approaches (Kundu et al, 1997;Maeda et al, 2000;Ishihama, 2000;Gruber & Gross, 2003;Paget & Helmann, 2003;Patten et al, 2004;Typas et al, 2007;Ades, 2008;Riordan et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families

Watanabe

Ishihama

2014

Microbiology

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The expression pattern of the genome in Escherichia coli is controlled by regulating the utilization of a limited number of RNA polymerases between a total of 4600 genes on its genome. The distribution pattern of RNA polymerase on the genome changes after two steps of protein-protein interaction with seven sigma subunits and about 300 transcription factors (TFs). Based on a systematic search for the regulation target promoters recognized by each TF, we propose two novel concepts: each TF regulates a number of target promoters; and each promoter is regulated by many TFs. In parallel, attempts have been made to determine the intracellular concentrations of all TFs using two systems: quantitative immunoblot analysis using TF-specific antibodies; and reporter assay of TF promoter activities. The direct measurement of TF protein level has so far been published for a set of 60 regulators with known functions. This study describes the determination of growth phase-dependent expression levels of 90 TFs using the reporter assay system. The translational fusion vector was constructed from the TF promoter sequence including an N-terminal proximal TF segment and the reporter GFP. At the beginning of cell growth, highlevel expression was observed only for a small number of TFs. In the exponential phase, approximately 80 % TFs are expressed, but the expressed TF species change upon transfer to the stationary phase. Significant changes in the pattern of TF expression were observed between aerobic and anaerobic conditions. The list of intracellular levels of TFs provides further understanding to the transcription regulation of the E. coli genome under various stressful conditions. INTRODUCTIONSingle-cell bacteria are directly exposed to frequently changing environments in nature and thus carry sophisticated genetic systems for adaptation to environmental changes (Ishihama, 2010(Ishihama, , 2012Yamamoto, 2014). On the basis of the complete genome sequence of several model Escherichia coli strains, the whole set of about 4600 genes has been predicted to exist in E. coli K-12 (Hayashi et al., 2006;Riley et al., 2006). In growing E. coli cells under laboratory culture conditions, only one-quarter to one-third of the genes on its genome are expressed, the others remaining silent. The majority of these uncharacterized silent genes must be expressed and utilized for adaptation and survival of E. coli under stressful conditions. Thus, even for this well-characterized model organism, the gene functions remain unidentified or unpredicted for approximately one-quarter because expression conditions of these silent genes have not yet been established.As the total number of RNA polymerases (RNAPs) in E. coli K-12 is less than the total number of genes on its genome (Ishihama, 2000), we proposed a model in which the change in genome transcription pattern takes place mainly through controlling the utilization of this limited number of transcription apparatuses between 4600 genes on the genome (Ishihama, 2010(Ishihama, , 2012. Two groups of reg...

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Two Extracytoplasmic Function Sigma Subunits, ς ^E and ς ^FecI , of Escherichia coli : Promoter Selectivity and Intracellular Levels

Abstract: The promoter selectivity of two extracytoplasmic function (ECF) subfamily subunits, E

Cited by 51 publications

References 36 publications

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families

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Two Extracytoplasmic Function Sigma Subunits, ς E and ς FecI , of Escherichia coli : Promoter Selectivity and Intracellular Levels

Abstract: The promoter selectivity of two extracytoplasmic function (ECF) subfamily subunits, E

Cited by 51 publications

References 36 publications

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families

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Two Extracytoplasmic Function Sigma Subunits, ς ^E and ς ^FecI , of Escherichia coli : Promoter Selectivity and Intracellular Levels