scholarly journals DNA sequence effects on single base deletions arising during DNA polymerization in vitro by Escherichia coli Klenow fragment polymerase.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 709-719
Author(s):  
F J Wang ◽  
L S Ripley
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Klenow Fragment ◽  
Single Base ◽  
Dna Polymerization ◽  
Sequence Effects ◽  
Frequent Mutations ◽  
Related Dnas ◽  
Dna Strand

Abstract Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent mutations were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.

scholarly journals Mutational Scanning and Affinity Cleavage Analysis of UhpA-Binding Sites in the Escherichia coli uhpT Promoter

2002 ◽  
Vol 184 (10) ◽  
pp. 2682-2691 ◽  
Author(s):  
Igor N. Olekhnovich ◽  
Robert J. Kadner
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Response Regulators ◽  
Binding Region ◽  
Nucleotide Substitutions ◽  
Iron Chelate ◽  
Affinity Cleavage

ABSTRACT UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from −80 to −50 and the weak-binding (W) region from −50 to −32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.

scholarly journals fleQ, the Gene Encoding the Major Flagellar Regulator of Pseudomonas aeruginosa, Is σ70 Dependent and Is Downregulated by Vfr, a Homolog of Escherichia coli Cyclic AMP Receptor Protein

2002 ◽  
Vol 184 (19) ◽  
pp. 5240-5250 ◽  
Author(s):  
Nandini Dasgupta ◽  
Evan P. Ferrell ◽  
Kristen J. Kanack ◽  
Susan E. H. West ◽  
Reuben Ramphal
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Receptor Protein ◽  
Regulatory Cascade

ABSTRACT The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a σ70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5′-AATTGACTAATCGTTCACATTTG-3′. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative −10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli σ70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this −10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the −10 region to initiate transcription.

Bespoke RNA recognition by Pumilios

2015 ◽  
Vol 43 (5) ◽  
pp. 801-806 ◽  
Author(s):  
Thomas A. Edwards
Keyword(s):  
Rna Binding ◽  
Consensus Sequence ◽  
Rna Binding Protein ◽  
Rna Recognition ◽  
Binding Mechanism ◽  
Recognition Sequence ◽  
Single Base ◽  
Puf Domain ◽  
Binding Sequence

Pumilio is an RNA-binding protein originally identified in Drosophila, with a Puf domain made up of eight Puf repeats, three helix bundles arranged in a rainbow architecture, where each repeat recognizes a single base of the RNA-binding sequence. The eight-base recognition sequence can therefore be modified simply via mutation of the repeat that recognizes the base to be changed and this is understood in detail via high-resolution crystal structures. The binding mechanism is also altered in a variety of homologues from different species, with bases flipped out from the binding site to regenerate a consensus sequence. Thus Pumilios can be designed with bespoke RNA recognition sequences and can be fused to nucleases, split GFP, etc. as tools in vitro and in cells.

scholarly journals PdhR (Pyruvate Dehydrogenase Complex Regulator) Controls the Respiratory Electron Transport System in Escherichia coli

2007 ◽  
Vol 189 (15) ◽  
pp. 5534-5541 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Yuji Ishida ◽  
Kayoko Yamada ◽  
Kaneyoshi Yamamoto ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Electron Transport ◽  
Transport System ◽  
Pyruvate Dehydrogenase ◽  
Fluorescent Protein ◽  
Pyruvate Dehydrogenase Complex ◽  
Consensus Sequence ◽  
Electron Transport System ◽  
Respiratory Electron Transport

ABSTRACT The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

scholarly journals Chemical roadblocking of DNA transcription for nascent RNA display

2020 ◽  
Vol 295 (19) ◽  
pp. 6401-6412
Author(s):  
Eric J. Strobel ◽  
John T. Lis ◽  
Julius B. Lucks
Keyword(s):  
Escherichia Coli ◽  
Rna Structure ◽  
Triethylene Glycol ◽  
Site Specific ◽  
Rna Molecules ◽  
Protein Factor ◽  
Nascent Rna ◽  
Dna Strand ◽  
And Function

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2′-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

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 An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence.

Genetics ◽  
1988 ◽  
Vol 118 (2) ◽  
pp. 181-191
Author(s):  
J G de Boer ◽  
L S Ripley
Keyword(s):  
Bacteriophage T4 ◽  
Consensus Sequence ◽  
Dna Repeats ◽  
Klenow Fragment ◽  
Purine Base ◽  
Vitro Assay ◽  
Reading Frame ◽  
Dna Primers

Abstract The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

scholarly journals The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2838-2844 ◽  
Author(s):  
Nicoletta Castiglione ◽  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Signal Molecules ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
E Coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

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