scholarly journals Selective Promoter Recognition by Chlamydial σ28 Holoenzyme

2006 ◽  
Vol 188 (21) ◽  
pp. 7364-7377 ◽  
Author(s):  
Li Shen ◽  
Xiaogeng Feng ◽  
Yuan Yuan ◽  
Xudong Luo ◽  
Thomas P. Hatch ◽  
...  
Keyword(s):  
Core Promoter ◽  
Sigma Factors ◽  
Promoter Sequences ◽  
E Coli ◽  
The Core ◽  
Recognition Specificity ◽  
Promoter Recognition ◽  
Transcription In Vitro

ABSTRACT The σ transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma factors, σ66, σ54, and σ28. We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis σ28 (σ28 Ct). One involved the arabinose-induced expression of plasmid-encoded σ28 Ct in a strain of Escherichia coli defective in the σ28 structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant σ28 Ct. These approaches were used to investigate the interactions of σ28 Ct with the σ28 Ct-dependent hctB promoter and selected E. coli σ28 (σ28 Ec)-dependent promoters, in parallel, compared with the promoter recognition properties of σ28 EC. Our results indicate that RNAP containing σ28 Ct has at least three characteristics: (i) it is capable of recognizing some but not all σ28 EC-dependent promoters; (ii) it can distinguish different promoter structures, preferentially activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than σ28 EC in recognizing variants with different spacing lengths separating the −35 and −10 elements of the core promoter.

scholarly journals Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

1998 ◽  
Vol 180 (20) ◽  
pp. 5375-5383 ◽  
Author(s):  
Wilma Ross ◽  
Sarah E. Aiyar ◽  
Julia Salomon ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Core Promoter ◽  
Consensus Sequence ◽  
Α Subunit ◽  
Core Promoters ◽  
E Coli ◽  
Promoter Recognition ◽  
Transcription In Vitro ◽  
Up Elements

ABSTRACT The α subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, λp R, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to thelac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.

scholarly journals Roles for Non-TATA Core Promoter Sequences in Transcription and Factor Binding

2000 ◽  
Vol 20 (10) ◽  
pp. 3608-3615 ◽  
Author(s):  
Branden S. Wolner ◽  
Jay D. Gralla
Keyword(s):  
Rna Polymerase Ii ◽  
Core Promoter ◽  
Core Region ◽  
Promoter Activation ◽  
Promoter Sequences ◽  
The Core ◽  
General Transcription Factors ◽  
Specific Effects ◽  
Transcription In Vitro

ABSTRACT Sequence blocks within the core region were swapped among RNA polymerase II promoters to explore effects on transcription in vitro. The pair of blocks flanking TATA strongly influenced general transcription, with an additional effect on promoter activation. These flanking elements induced a change in the ratio of activated to basal transcription, whereas swapping TATA and initiator sequences only altered general transcription levels. Swapping the flanking blocks influenced binding by general transcription factors TBP and TFIIB. The results suggest that the architecture of the extended core sequence is important in determining promoter-specific effects on both general transcription levels and the tightness of regulation.

scholarly journals Analysis of Core Promoter Sequences Located Downstream from the TATA Element in the hsp70 Promoter from Drosophila melanogaster

2001 ◽  
Vol 21 (5) ◽  
pp. 1593-1602 ◽  
Author(s):  
Chwen-Huey Wu ◽  
Lakshmi Madabusi ◽  
Hokuto Nishioka ◽  
Peter Emanuel ◽  
Michael Sypes ◽  
...  
Keyword(s):  
Core Promoter ◽  
P Element ◽  
Multiple Sequence ◽  
Promoter Sequences ◽  
Tata Element ◽  
Sequence Elements ◽  
Cross Links ◽  
Transcription In Vitro

ABSTRACT TFIID recognizes multiple sequence elements in thehsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

scholarly journals Efficient and Specific Initiation of Subgenomic RNA Synthesis by Cucumber Mosaic Virus Replicase In Vitro Requires an Upstream RNA Stem-Loop

2000 ◽  
Vol 74 (23) ◽  
pp. 11201-11209 ◽  
Author(s):  
M.-H. Chen ◽  
M. J. Roossinck ◽  
C. C. Kao
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Stem Loop ◽  
Promoter Sequences ◽  
Promoter Recognition

ABSTRACT We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions −28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions −28 to −5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clemens Höflich ◽  
Angela Brieger ◽  
Stefan Zeuzem ◽  
Guido Plotz
Keyword(s):  
Wilson Disease ◽  
Transcription Initiation ◽  
Transcriptional Activity ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Copper Metabolism ◽  
Biologically Relevant ◽  
The Core ◽  
Translational Start

AbstractPathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

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