Location of essential sequence elements at the Escherichia coli melAB promoter.
Ontology highlight
ABSTRACT:
SUBMITTER: Keen J
PROVIDER: S-EPMC1217641 | biostudies-other | 1996 Sep
REPOSITORIES: biostudies-other
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Project description:RNA polymerase of both bacteria and eukaryotes can stall or pause within tens of base pairs of its initiation site at the promoter, a state that may reflect important regulatory events in early transcription. In the bacterial model system, the ?(70) initiation factor stabilizes such pauses by binding a downstream repeat of a promoter segment, especially the '-10' promoter element. We first show here that the '-35' promoter element also can stabilize promoter-proximal pausing, through interaction with ?(70) region 4. We further show that an essential element of either type of pause is a sequence just upstream of the site of pausing that stabilizes RNA polymerase backtracking. Although the pause is not intrinsically backtracked, we suggest that the same sequence element is required both to stabilize the paused state and to potentiate backtracking.
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Project description:BACKGROUND: Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome. RESULTS: Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences. CONCLUSIONS: This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.
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Project description:The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.
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Project description:Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we experimentally mined novel constitutive promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Through the expression profiling of fluorescent clones from two independent soil sample libraries, we have analyzed the regulatory dynamics of 260 clones with candidate promoters as a set of active metagenomic promoters in the host Escherichia coli. Through an in-depth analysis of selected clones, we were able to further explore the architecture of metagenomic fragments and to report the presence of multiple promoters per fragment with a dominant promoter driving the expression profile. These approaches resulted in the identification of 33 novel active promoters from metagenomic DNA originated from very diverse phylogenetic groups. The in silico and in vivo analysis of these individual promoters allowed the generation of a constitutive promoter consensus for exogenous sequences recognizable by E. coli in metagenomic studies. The results presented here demonstrates the potential of functional metagenomics for exploring environmental bacterial communities as a source of novel regulatory genetic parts to expand the toolbox for microbial engineering.
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Project description:The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.
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Project description:Classical elements of σ(70) bacterial promoters include the -35 element ((-35)TTGACA(-30)), the -10 element ((-12)TATAAT(-7)), and the extended -10 element ((-15)TG(-14)). Although the -35 element, the extended -10 element, and the upstream-most base in the -10 element ((-12)T) interact with σ(70) in double-stranded DNA (dsDNA) form, the downstream bases in the -10 motif ((-11)ATAAT(-7)) are responsible for σ(70)-single-stranded DNA (ssDNA) interactions. In order to directly reflect this correspondence, an extension of the extended -10 element to a so-called -15 element ((-15)TGnT(-12)) has been recently proposed. I investigated here the sequence specificity of the proposed -15 element and its relationship to other promoter elements. I found a previously undetected significant conservation of (-13)G and a high degeneracy at (-15)T. I therefore defined the -15 element as a degenerate motif, which, together with the conserved stretch of sequence between -15 and -12, allows treating this element analogously to -35 and -10 elements. Furthermore, the strength of the -15 element inversely correlates with the strengths of the -35 element and -10 element, whereas no such complementation between other promoter elements was found. Despite the direct involvement of -15 element in σ(70)-dsDNA interactions, I found a significantly stronger tendency of this element to complement weak -10 elements that are involved in σ(70)-ssDNA interactions. This finding is in contrast to the established view, according to which the -15 element provides a sufficient number of σ(70)-dsDNA interactions, and suggests that the main parameter determining a functional promoter is the overall promoter strength.
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Project description:Using a novel multiplexed reporter assay, we characterize promoter activity of hundreds of thousands of DNA sequences spanning the entire E. coli genome. We use this powerful assay to identify promoters throughout the E. coli genome and systematically dissect their regulatory motifs which encode promoter activity using a series of experiments.
2020-06-01 | GSE144621 | GEO
Project description:Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded Ecoli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.
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