scholarly journals The loopometer: a quantitative in vivo assay for DNA-looping proteins

2021 ◽  
Author(s):  
Nan Hao ◽  
Adrienne E Sullivan ◽  
Keith E Shearwin ◽  
Ian B Dodd
Keyword(s):  
Binding Sites ◽  
Dna Looping ◽  
Lacz Reporter Gene ◽  
Effective Distance ◽  
E Coli ◽  
In Vivo Assay ◽  
Reporter Gene Analysis ◽  
Phage Proteins ◽  
Laci Repressor

Abstract Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.

scholarly journals Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1779-1788 ◽  
Author(s):  
Graham P. Stafford ◽  
Tomoo Ogi ◽  
Colin Hughes
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Class Ii ◽  
Promoter Regions ◽  
E Coli ◽  
Flagellar Genes

The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD2C2. To assess the extent to which FlhD2C2 directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42–44 bp inverted repeat FlhD2C2 binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD2C2 binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD2C2 over the K D range 38–356 nM, and of the nine corresponding chromosomal promoter–lacZ fusions, those of the four genes b1904, b2446, wzz fepE and gltI showed up to 50-fold dependence on FlhD2C2 in vivo. In comparison, four representative flagella class II promoters bound FlhD2C2 in the K D range 12–43 nM and were upregulated in vivo 30- to 990-fold. The FlhD2C2-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted −35 motif of the FlhD2C2-activated σ 70 promoters, as is the case with FlhD2C2-dependent class II flagellar promoters. The data indicate a wider FlhD2C2 regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.

scholarly journals High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli

2015 ◽  
Vol 112 (23) ◽  
pp. 7177-7182 ◽  
Author(s):  
Nicole A. Becker ◽  
L. James Maher
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Dna Looping ◽  
Chromosomal Protein ◽  
Bent Dna ◽  
E Coli ◽  
Dna Loop ◽  
Eukaryotic Chromatin ◽  
Negative Supercoiling

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

scholarly journals The Role of Gcr1p in the Transcriptional Activation of Glycolytic Genes in Yeast Saccharomyces cerevisiae

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 521-532 ◽  
Author(s):  
Hiroshi Uemura ◽  
Miho Koshio ◽  
Yoko Inoue ◽  
M Cecilia Lopez ◽  
Henry V Baker
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Regulatory Sequences ◽  
Lacz Reporter ◽  
Lacz Reporter Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Glycolytic Gene ◽  
Synthetic Oligonucleotides

To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem CT-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5′ noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants had single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.

scholarly journals Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12

2021 ◽  
Vol 12 ◽  
Author(s):  
Tomohiro Shimada ◽  
Hiroshi Ogasawara ◽  
Ikki Kobayashi ◽  
Naoki Kobayashi ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Binding Sites ◽  
Target Genes ◽  
Minority Group ◽  
E Coli ◽  
Single Target ◽  
K 12

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

scholarly journals A- and T-Tract-Mediated Intrinsic Curvature in Native DNA between the Binding Site of the Upstream Activator NtrC and the nifLA Promoter of Klebsiella pneumoniaeFacilitates Transcription

1999 ◽  
Vol 181 (17) ◽  
pp. 5296-5302 ◽  
Author(s):  
Amrita Kaur Cheema ◽  
Nirupam Roy Choudhury ◽  
H. K. Das
Keyword(s):  
Integration Host Factor ◽  
Dna Looping ◽  
Dna Curvature ◽  
Intrinsic Curvature ◽  
E Coli ◽  
Dna Base ◽  
Dna Fragment ◽  
The One ◽  
Rna Polymerase Holoenzyme

ABSTRACT The nif promoters of Klebsiella pneumoniaemust be activated by proteins bound to upstream sequences which are thought to interact with the ς54-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the nifLA promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the Escherichia coli lacZ gene of the DNA fragment containing the nifLA promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in K. pneumoniae and E. coliwas monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from thenifLA promoter of K. pneumoniae.

scholarly journals Self-Assembly of Tail Tube Protein of Bacteriophage vB_EcoS_NBD2 into Extremely Long Polytubes in E. coli and S. cerevisiae

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 208 ◽  
Author(s):  
Aliona Špakova ◽  
Eugenijus Šimoliūnas ◽  
Raminta Batiuškaitė ◽  
Simonas Pajeda ◽  
Rolandas Meškys ◽  
...  
Keyword(s):  
Self Assembly ◽  
Tail Length ◽  
Cell Protein ◽  
Tubular Structures ◽  
E Coli ◽  
Physical Conditions ◽  
Self Assembling ◽  
Phage Proteins ◽  
Purification Protocol

Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%–90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and 0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.

The expression ofnifBgene fromHerbaspirillum seropedicaeis dependent upon the NifA and RpoN proteins

2006 ◽  
Vol 52 (12) ◽  
pp. 1199-1207 ◽  
Author(s):  
Fabiane G.M Rego ◽  
Fábio O Pedrosa ◽  
Leda S Chubatsu ◽  
M Geoffrey Yates ◽  
Roseli Wassem ◽  
...  
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Integration Host Factor ◽  
Lacz Fusion ◽  
Herbaspirillum Seropedicae ◽  
Band Shift ◽  
E Coli ◽  
Low Levels ◽  
Ihf Protein

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a –24/–12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.Key words: Herbaspirillum seropedicae, nif, nitrogen fixation, NifA, RpoN.

scholarly journals Superimposition of TyrR Protein-Mediated Regulation on Osmoresponsive Transcription of Escherichia coli proUIn Vivo

1998 ◽  
Vol 180 (24) ◽  
pp. 6743-6748 ◽  
Author(s):  
J. Gowrishankar ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Osmotic Regulation ◽  
E Coli ◽  
Regulator Protein ◽  
The Creation

ABSTRACT Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the ς70-controlled promoter of proU inEscherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by l-phenylalanine (Phe), as well as repression by l-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli.

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