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.

scholarly journals Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ28 RNA Polymerase

2006 ◽  
Vol 188 (15) ◽  
pp. 5524-5531 ◽  
Author(s):  
Hilda Hiu Yin Yu ◽  
Elizabeth G. Di Russo ◽  
Megan A. Rounds ◽  
Ming Tan
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
Promoter Sequence ◽  
Promoter Element ◽  
Developmental Gene ◽  
Spacer Length ◽  
E Coli ◽  
Promoter Recognition ◽  
Developmental Gene Regulation

ABSTRACT σ28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial σ28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the σ28-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a −35 element recognized by chlamydial σ28 RNA polymerase that resembles the consensus −35 sequence. Within the −10 element, however, chlamydial σ28 RNA polymerase showed a striking preference for a CGA sequence at positions −12 to −10 rather than the longer consensus −10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli σ28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the −10 promoter element recognized by σ28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that σ28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for σ28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred σ28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n11/12-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n11/12 is a spacer of 11 or 12 nt.

scholarly journals Synthetic Promoter Designs Enabled by a Comprehensive Analysis of Plant Core Promoters

2021 ◽  
Author(s):  
Tobias Jores ◽  
Jackson Tonnies ◽  
Travis Wrightsman ◽  
Edward S. Buckler ◽  
Josh Cuperus ◽  
...  
Keyword(s):  
Computational Models ◽  
Core Promoter ◽  
Gc Content ◽  
Regulatory Elements ◽  
Great Promise ◽  
35S Promoter ◽  
Promoter Strength ◽  
Promoter Elements ◽  
Native Promoter ◽  
Highly Active

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements - notably the TATA-box - as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detected species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

scholarly journals Identification of cis-Active Elements in Ustilago maydis mig2 Promoters Conferring High-Level Activity During Pathogenic Growth in Maize

2005 ◽  
Vol 18 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Jan W. Farfsing ◽  
Kathrin Auffarth ◽  
Christoph W. Basse
Keyword(s):  
Expression Profile ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Ustilago Maydis ◽  
Tumor Formation ◽  
Homologous Gene ◽  
Specific Expression ◽  
High Level

The Ustilago maydis mig2 cluster comprises five highly homologous genes that display a pronounced plant-specific expression profile. A 350-bp mig2-5 promoter fragment contained all elements sufficient to confer differential promoter activity. Mutational analysis of this region, fused to the green fluorescent protein reporter gene, allowed dissecting core promoter elements required for high-level promoter activity from elements conferring inducible expression in planta. In particular, the presence of several 5′-CCA-3′ motifs within a short stretch of the mig2-5 promoter was decisive for inducible promoter activity. On this basis, we reconstituted an artificial promoter whose inducible activity specifically relied on multiple CCA motifs. In addition, we identified a novel mig2 homologous gene, mig2-6, that is not part of the mig2 cluster, but displayed the strongest differential expression profile among mig2 genes. The deletion of all six mig2 genes did not compromise the ability to induce tumor formation in infected maize plants. Comparative sequence analysis including the mig2-6 promoter revealed an over-representation of the consensus motif 5′-MNMNWNCCAMM-3′. We discuss putative transcriptional activators involved in mig2 regulation.

scholarly journals DNA Unwinding Mechanism for the Transcriptional Activation ofmomP1Promoter by the Transactivator Protein C of Bacteriophage Mu

2001 ◽  
Vol 276 (50) ◽  
pp. 46941-46945 ◽  
Author(s):  
Shashwati Basak ◽  
Valakunja Nagaraja
Keyword(s):  
Conformational Changes ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Transcription Activation ◽  
Bacteriophage Mu ◽  
Promoter Elements ◽  
Spacer Length ◽  
C Protein ◽  
Transactivator Protein ◽  
Reduced Activity

Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation. C protein of bacteriophage Mu appears to transactivate themomgene of the phage by this mode. DNA binding by C to its site leads to torsional changes that seem to compensate for a weakmomP1 promoter having a suboptimal spacing of 19 bp between the poor −35 and −10 elements. The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter. In this study, the model has been tested by mutational analysis of the spacer region ofmomP1 and by assessing the strength of the mutant promoters. The response to C-mediated transactivation was dependent on the spacer length of the promoters. Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level. A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model. The results imply a role for C-mediated unwinding inmomtranscription activation.

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 More than a Spacer: The Bacterial Promoter Spacer Modulates Promoter Strength and Timing by Length, Sequence, TG-Motifs and DNA Supercoiling Sensitivity

2021 ◽  
Author(s):  
Carlo A. Klein ◽  
Marc Teufel ◽  
Carl J. Weile ◽  
Patrick Sobetzko
Keyword(s):  
Promoter Activity ◽  
Gc Content ◽  
Promoter Sequence ◽  
Dna Supercoiling ◽  
Promoter Strength ◽  
Time Resolved ◽  
Spacer Length ◽  
Sequence Composition ◽  
Promoter Architecture ◽  
Transcriptomics Data

Abstract Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals a, RNAP binding enhancing but expression damping, extension to the –10 region. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.

scholarly journals Characterization of the mouse dynamin I gene promoter and identification of sequences that direct expression in neuronal cells

2000 ◽  
Vol 351 (3) ◽  
pp. 661-668 ◽  
Author(s):  
Jiyun YOO ◽  
Sang Seop LEE ◽  
Moon-Jin JEONG ◽  
Kyung Im LEE ◽  
Byoung-Mog KWON ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Gc Content ◽  
Proximal Region ◽  
Minimal Promoter ◽  
Binding Motif ◽  
Mobility Shift ◽  
Flanking Region ◽  
Dynamin I ◽  
I Gene

Dynamin I is expressed at high levels in brain and its expression is regulated during the developmental stages of brain. To elucidate the molecular mechanism by which the expression is tissue-specifically regulated, we cloned the 5′-flanking region of the mouse dynamin I gene and determined the nucleotide sequence of 1036 bases upstream from the translation start site. Transient transfection studies with a chloramphenicol acetyltransferase reporter gene in neuroblastoma NS20Y and Lewis lung cells demonstrated that the 5′-flanking region has a cell-type-specific promoter activity. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 195bp upstream of the translation initiation codon (-90 to +105). The minimal promoter was embedded in a GC-rich region (75% GC content), in which an Sp1-binding motif and a nuclear factor (NF)-κB-like element (NE-1) were found, but it lacked TATA and CAAT boxes. Mutational analysis and electrophoretic mobility-shift assay analysis revealed that Sp1 binds to the Sp1 site and that this element is critical for the promoter activity of the dynamin I gene. We found that the NE-1 sequence is required for the expression of the dynamin I gene but NEBP (NE-1-binding protein), which binds to the NE-1 sequence, is not NF-κB. We also found that one base in the NE-1 sequence (the underlined G residue in GGGATTCG̲CGGA) is critical for binding specificity to discriminate between NEBP and NF-κB. By UV cross-linking analysis, we found that NEBP is an approx. 104kDa nuclear protein.

scholarly journals Promoter prediction in the rhizobia

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1751-1763 ◽  
Author(s):  
Shawn R. MacLellan ◽  
Allyson M. MacLean ◽  
Turlough M. Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Mutational Analysis ◽  
Background Activity ◽  
Trna Genes ◽  
Expression Data ◽  
Ribosomal Protein Genes ◽  
Significant Similarity ◽  
Promoter Sequences ◽  
E Coli ◽  
Experimental Identification

The ability to recognize and predict non-σ 54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the −35 and −10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli σ 70 promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.

scholarly journals Major Histocompatibility Complex Class I Core Promoter Elements Are Not Essential for Transcriptionin vivo

2013 ◽  
Vol 33 (22) ◽  
pp. 4395-4407 ◽  
Author(s):  
Zohar S. Barbash ◽  
Jocelyn D. Weissman ◽  
John A. Campbell ◽  
Jie Mu ◽  
Dinah S. Singer
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Transgene Expression ◽  
Promoter Activity ◽  
Core Promoter ◽  
Class I ◽  
Complex Class ◽  
Promoter Elements ◽  
Tissue Specific ◽  
Histocompatibility Complex

The role of core promoter elements in regulating transcription initiation is largely unknown for genes subject to complex regulation. Major histocompatibility complex class I genes are ubiquitously expressed and governed by tissue-specific and hormonal signals. Transcription initiates at multiple sites within the core promoter, which contains elements homologous to the canonical elements CCAAT, TATAA, Sp1 binding site (Sp1BS), and Initiator (Inr). To determine their functions, expression of class I transgenes with individually mutated elements was assessed. Surprisingly, all mutant promoters supported transcription. However, each mutated core promoter element had a distinct effect on expression: CAAT box mutations modulated constitutive expression in nonlymphoid tissues, whereas TATAA-like element mutations dysregulated transcription in lymphoid tissues. Inr mutations aberrantly elevated expression. Sp1BS element mutations resulted in variegated transgene expression. RNA polymerase II binding and histone H3K4me3 patterns correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the wild-type, TATAA-like, and CAAT mutant promoters were activated by gamma interferon, the Sp1 and Inr mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each plays a distinct role in promoter activity, chromatin structure, tissue-specific expression, and extracellular signaling.

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