scholarly journals C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

2021 ◽  
Author(s):  
Christopher Schubert ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Concerted Action ◽  
Phosphotransferase System ◽  
Regulatory Pathways ◽  
E Coli ◽  
Fumarate Respiration ◽  
Genes Encoding ◽  
Two Hybrid

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation. The aerobic C4-DC transporter DctA interacted with the regulator EIIAGlc of the E. coli glucose phosphotransferase system. It is suggested that EIIAGlc inhibits C4-DC uptake by DctA in the presence of the preferred substrate glucose.

scholarly journals Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

2020 ◽  
Vol 295 (46) ◽  
pp. 15454-15463 ◽  
Author(s):  
Chelsey R. Fontenot ◽  
Homyra Tasnim ◽  
Kathryn A. Valdes ◽  
Codrina V. Popescu ◽  
Huangen Ding
Keyword(s):  
Escherichia Coli ◽  
Iron Content ◽  
Iron Homeostasis ◽  
Ferric Uptake Regulator ◽  
Intracellular Iron ◽  
Free Iron ◽  
E Coli ◽  
Genes Encoding ◽  
Fur Protein ◽  
Mutant Cells

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron–sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

scholarly journals Detection and Quantification of Superoxide Formed within the Periplasm of Escherichia coli

2006 ◽  
Vol 188 (17) ◽  
pp. 6326-6334 ◽  
Author(s):  
Sergei Korshunov ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Phagocytic Cells ◽  
Free Living ◽  
E Coli ◽  
Superoxide Formation ◽  
Genes Encoding ◽  
Copper Zinc ◽  
Primary Substrate

ABSTRACT Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, including Escherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures of E. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm of E. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Altered Utilization of N-Acetyl-d-Galactosamine by Escherichia coli O157:H7 from the 2006 Spinach Outbreak

2007 ◽  
Vol 190 (5) ◽  
pp. 1710-1717 ◽  
Author(s):  
Amit Mukherjee ◽  
Mark K. Mammel ◽  
J. Eugene LeClerc ◽  
Thomas A. Cebula
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Culture Collection ◽  
Base Substitution ◽  
Phosphotransferase System ◽  
Single Nucleotide ◽  
E Coli ◽  
Single Nucleotide Change ◽  
Phenotype Comparison ◽  
In Silico Analyses

ABSTRACT In silico analyses of previously sequenced strains of Escherichia coli O157:H7, EDL933 and Sakai, localized the gene cluster for the utilization of N-acetyl-d-galactosamine (Aga) and d-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)Aga/Gam, EIIBAga, EIICAga, and EIIDAga of the Aga PTS are present, E. coli O157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIICGam, and consequently, E. coli O157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates of E. coli O157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+ Gam− phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associated E. coli O157:H7 outbreak, were analyzed, all (194/194) displayed an Aga− Gam− phenotype. Comparison of aga/gam sequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C→A:T) in the agaF gene in the spinach-associated isolates. The base substitution in agaF, which encodes EIIAAga/Gam of the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associated E. coli O157:H7 isolates harbored this same G:C→A:T substitution. Notably, when agaF + was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga− phenotype in these isolates.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σEin Escherichia coli

2015 ◽  
Vol 197 (14) ◽  
pp. 2316-2324 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Ectopic Expression ◽  
Growth Media ◽  
Content Type ◽  
E Coli ◽  
Envelope Stress ◽  
Genes Encoding ◽  
Σ Factor ◽  
Mutant Forms

ABSTRACTσE, an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth ofEscherichia colinot only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of thehicBgene has been reported to suppress the lethality caused by the loss of σE.hicBencodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σEand, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption ofhicAabolished suppression of the σEessentiality in the absence ofhicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σEessentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins,E. colicells with no suppressor mutations grew independently of σE. Taken together, our results indicate that the activation of TA system toxins can suppress the σEessentiality and affect the extracytoplasmic stress responses.IMPORTANCEσEis an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σEis indispensable for the survival ofE. colieven under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σEessentiality, suggesting a connection between TA systems and σEfunction. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

scholarly journals High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

2005 ◽  
Vol 71 (12) ◽  
pp. 8157-8164 ◽  
Author(s):  
Ying Zhong Tang ◽  
Karina Yew Hoong Gin ◽  
Tok Hoon Lim
Keyword(s):  
Escherichia Coli ◽  
Signal Intensity ◽  
Fluorescence Signal ◽  
Standard Protocol ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Hybridization Efficiency ◽  
Hybridization Time ◽  
Seawater Samples

ABSTRACT Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

scholarly journals Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Yingbo Shen ◽  
Zuowei Wu ◽  
Yang Wang ◽  
Rong Zhang ◽  
Hong-Wei Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Fluoroquinolone Resistance ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

scholarly journals The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

2006 ◽  
Vol 188 (4) ◽  
pp. 1279-1285 ◽  
Author(s):  
Deborah M. Hinton ◽  
Srilatha Vuthoori ◽  
Rebecca Mulamba
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Dynamic Equilibrium ◽  
Bacteriophage T4 ◽  
Direct Interaction ◽  
Binding Properties ◽  
E Coli ◽  
Dna Binding Properties ◽  
Two Hybrid

ABSTRACT The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P uvsX . However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70 plus core, yielding more free σ70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70 arises through an indirect effect.

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