Intracellular Concentrations of 65 Species of Transcription Factors with Known Regulatory Functions in Escherichia coli

Abstract

The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

“…3, 0 nM data). In vivo, there is a high concentration of DNA-binding protein, and in particular the total concentration of Fis in a rapidly growing E. coli cell is roughly 30 to 50 M (14,18,19), suggesting that the free Fis concentration should be at least as high as in the range of a few hundred nanomoles per liter. Notably, for GFP-Fis in the presence of WT Fis in solution, a 40-s binding lifetime is obtained for about 400 nM WT Fis (Fig.…”

“…The exchange rate constant (k exch ) was found to be 6 ϫ 10 4 M Ϫ1 s Ϫ1 , describing the increase of off-rate from the zero-concentration level (k off,0 ) of Ϸ1 ϫ 10 Ϫ3 s Ϫ1 to about four times that amount at a bulk Fis concentration of 50 nM (5). Fis concentrations in vivo vary widely with growth conditions but are in the 10 to 50 M range during fast growth (18,19), suggesting an unbound Fis concentration of at least 100 nM, sufficient to strongly increase the Fis off-rate from its zero-concentration value. Furthermore, given the possibility for facilitated dissociation to be driven by other species of protein (e.g., by HU as observed in vitro [5]), the off-rate of Fis or other DNA-binding proteins may be even more strongly perturbed in vivo.…”

“…The exchange rate constant (k exch ) was found to be 6 ϫ 10 4 M Ϫ1 s Ϫ1 , describing the increase of off-rate from the zero-concentration level (k off,0 ) of Ϸ1 ϫ 10 Ϫ3 s Ϫ1 to about four times that amount at a bulk Fis concentration of 50 nM (5). Fis concentrations in vivo vary widely with growth conditions but are in the 10 to 50 M range during fast growth (18,19), suggesting an unbound Fis concentration of at least 100 nM, sufficient to strongly …”

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

“…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.…”

“…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. The first report of results using the quantitative immunoblot system has been published, which describes the intracellular concentration of 60 species of TFs with known regulatory functions (Ishihama et al, 2014). As a quick and accurate means of monitoring the expression level of a number of TFs in E. coli cells under various growth conditions, we employed in this study the second method, in which the expression level of TFs was determined using a modified version of the promoter assay vector (Shimada et al, 2004).…”