Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

ABSTRACTTheatzS-atzT-atzU-atzV-atzWgene cluster of thePseudomonassp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream fromatzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operonatzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzRpromoter to form theatzRSTUVWoperon. A second, stronger promoter, PatzT, is found withinatzSand directs transcription of the four distal genes. PatzTis σNdependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination ofin vivomutational analysis and primer extension allowed us to locate the PatzTpromoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along within vivoexpression studies andin vitrobinding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzTtranscription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process.

Download Full-text

Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2777 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2777-2786 ◽

Cited By ~ 24

Author(s):

V Gailus-Durner ◽

J Xie ◽

C Chintamaneni ◽

A K Vershon

Keyword(s):

Transcriptional Activation ◽

Mutational Analysis ◽

Consensus Sequence ◽

Promoter Sequence ◽

Regulatory Elements ◽

Specific Gene ◽

Specific Expression ◽

Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

Download Full-text

Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.00404-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Mutational Analysis ◽

Wild Type ◽

Content Type ◽

Growth Defects ◽

C Terminus ◽

K 12 ◽

And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

Download Full-text

Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility

Biochemical Journal ◽

10.1042/bj20050874 ◽

2005 ◽

Vol 392 (1) ◽

pp. 241-248 ◽

Cited By ~ 92

Author(s):

Olivier Loudig ◽

Glenn A. Maclean ◽

Naomi L. Dore ◽

Luong Luu ◽

Martin Petkovich

Keyword(s):

Retinoic Acid ◽

Mutational Analysis ◽

Transcriptional Start Site ◽

Critical Role ◽

Transcriptional Response ◽

Luciferase Reporter ◽

Maximal Response ◽

Fold Induction

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483–1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10−8 M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARγ (RA receptor γ)/RXRα (retinoid X receptor α) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

Download Full-text

Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

10.1101/092445 ◽

2016 ◽

Cited By ~ 2

Author(s):

Molly Gasperini ◽

Gregory M. Findlay ◽

Aaron McKenna ◽

Jennifer H. Milbank ◽

Choli Lee ◽

...

Keyword(s):

Large Scale ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Mendelian Disease ◽

Regulatory Sequences ◽

Coding Region ◽

Endogenous Gene ◽

Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

Download Full-text

The MCP silencer of theDrosophila Abd-Bgene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Development ◽

10.1242/dev.128.11.2163 ◽

2001 ◽

Vol 128 (11) ◽

pp. 2163-2173 ◽

Cited By ~ 7

Author(s):

Ana Busturia ◽

Alan Lloyd ◽

Fernando Bejarano ◽

Michael Zavortink ◽

Hua Xin ◽

...

Keyword(s):

Reporter Gene ◽

Binding Sites ◽

Mutational Analysis ◽

Regulatory Elements ◽

Homeotic Gene ◽

Gaga Factor ◽

Polycomb Response Elements ◽

Silencer Element

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.

Download Full-text

A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5024-5032.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5024-5032

Author(s):

R Bassel-Duby ◽

M D Hernandez ◽

M A Gonzalez ◽

J K Krueger ◽

R S Williams

Keyword(s):

Mammalian Cells ◽

Mutational Analysis ◽

Transcriptional Start Site ◽

Upstream Sequence ◽

Specific Expression ◽

Sequence Element ◽

Promoter Function ◽

Human Myoglobin

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

Download Full-text

In vitro efficacy of transferrin-toxin conjugates against glioblastoma multiforme

Journal of Neurosurgery ◽

10.3171/jns.1992.76.5.0838 ◽

1992 ◽

Vol 76 (5) ◽

pp. 838-844 ◽

Cited By ~ 36

Author(s):

Walter A. Hall ◽

Aslak Godal ◽

Siri Juell ◽

Øystein Fodstad

Keyword(s):

Glioblastoma Multiforme ◽

Cytotoxic Activity ◽

Cell Lines ◽

Receptor Expression ◽

Binding Assays ◽

Content Type ◽

Monensin A ◽

Diferric Transferrin

✓ The cytotoxic activity of immunotoxins constructed with human diferric transferrin (Tfn) as the carrier ligand and an abrin variant Pseudomonas exotoxin A (PE) and the diphtheria toxin mutant cross-reacting material (CRM) 107 as the toxin moieties were studied in vitro. Three malignant human cell lines, the glioblastomas multiforme SNB19 and SF295 and the LOX melanoma, and a nonhuman control murine melanoma cell line B16 were assessed. The presence of transferrin receptors on the cell lines was confirmed by direct 125I-Tfn binding assays. The 50% protein synthesis inhibitory concentration (IC50) values for all cell lines demonstrated that Tfn-abrin variant and Tfn-PE had comparable potency and were both more effective than Tfn-CRM 107. Monensin, a carboxylic ionophore, potentiated the effect of Tfn-abrin variant against glioma cells approximately 35-fold with IC50 values of 4.0 × 10−13 M and 4.7 × 10−12 M for SNB19 and SF295, respectively. Cytotoxic activity of Tfn-abrin variant (with or without monensin) and Tfn-PE was correlated with the degree of Tfn receptor expression measured on the cell lines. The exquisite in vitro cytotoxicity of Tfn-abrin variant and Tfn-PE immunotoxins against glioma and melanoma cells warrants further in vivo evaluation and future consideration of these agents for potential clinical application against glioblastoma multiforme and leptomeningeal neoplasia.

Download Full-text

Control of the Arabinose Regulon in Bacillus subtilisby AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping

Journal of Bacteriology ◽

10.1128/jb.183.14.4190-4201.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4190-4201 ◽

Cited By ~ 23

Author(s):

Luı́s Jaime Mota ◽

Leonor Morais Sarmento ◽

Isabel de Sá-Nogueira

Keyword(s):

Regulatory Gene ◽

Regulatory Elements ◽

In Vitro Transcription ◽

Dna Looping ◽

Major Protein ◽

Flexible Control ◽

Transcription System ◽

Single Operator

ABSTRACT The proteins involved in the utilization of l-arabinose by Bacillus subtilis are encoded by thearaABDLMNPQ-abfA metabolic operon and by thearaE/araR divergent unit. Transcription from the ara operon, araE transport gene, andaraR regulatory gene is induced by l-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within thearaABDLMNPQ-abfA (ORA1 and ORA2) and araE (ORE1 and ORE2) promoters and noncooperatively to a single operator in the araR (ORR3) promoter region. Here, we have investigated how AraR controls transcription from theara regulon in vivo. A deletion analysis of theara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, ORE1-ORE2 and ORR3 are auxiliary operators for the autoregulation ofaraR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from thearaABDLMNPQ-abfA operon and araEpromoters, strongly suggesting that it is the major protein involved in the repression mechanism of l-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.

Download Full-text

Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

Journal of Bacteriology ◽

10.1128/jb.00690-16 ◽

2016 ◽

Vol 199 (5) ◽

Cited By ~ 3

Author(s):

Jennifer R. Tanner ◽

Palak G. Patel ◽

Jacqueline R. Hellinga ◽

Lynda J. Donald ◽

Celine Jimenez ◽

...

Keyword(s):

Legionella Pneumophila ◽

Mutational Analysis ◽

Transcriptional Regulator ◽

Intracellular Growth ◽

Content Type ◽

Expression Control ◽

Two Component ◽

Legionnaires Disease

ABSTRACT Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyRLp) is an ortholog of Escherichia coli OxyR; however, OxyRLp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila. Lack of OxyRLp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyRLp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyRLp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyRLp directly bound to these promoter regions, suggesting that OxyRLp is a direct repressor. Interestingly, a strain overexpressing OxyRLp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyRLp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein. IMPORTANCE Legionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyRLp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyRLp expression is regulated remain elusive, this study shows that OxyRLp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

Download Full-text