Escherichia coli K-12
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA260531 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1570164.fastq.gz | Fastqsanger.gz | |||
| SRR1570165.fastq.gz | Fastqsanger.gz | |||
| SRR1570166.fastq.gz | Fastqsanger.gz | |||
| SRR1570167.fastq.gz | Fastqsanger.gz |
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Project description:In this work we introduce a new high-throughput method, Spec-seq, that directly measures specificity by sequencing. It has several advantages over existing methods to quantify large collections (thousands) of binding site energies in one experiment. Using lac repressor as an example, we show that this method has excellent reproducibility giving energy measurements generally consistent within about 0.1kT. While not measuring in parallel as many different sequences as some of the higher throughput methods, we obtain high accuracies because we measure exactly what is necessary, the relative affinity to a large collection of sequences, without any mathematical fitting procedures or approximations required. It is similar to MITOMI in the number of different sites that can be analyzed in parallel, but it is much simpler to perform, requiring only a means to separate bound and unbound fractions which are then sequenced using standard high throughput, short read sequencing machines that are now readily available. When applied to the lac repressor we obtain, in a single experiment, data covering a large fraction of all the previous studies, plus thousands of additional variants, allowing us to compile a much more comprehensive profile of its specificity. We learn that the lac repressor can bind to sites of different lengths but that the preferred sequence, and the mode of binding, depends on the length. We also apply the method to two other members of the LacI/GalR protein family, PurR and YcjW, to obtain extensive models of their specificity and test the generality of the lac repressor's ability to bind to operators of variable length. We find that the lac repressor is apparently unique in its ability to bind with high affinity to sites of different lengths and with different modes of binding. 4 independent experiments.
2014-10-01 | E-GEOD-61223 | biostudies-arrayexpress
Project description:In this work we introduce a new high-throughput method, Spec-seq, that directly measures specificity by sequencing. It has several advantages over existing methods to quantify large collections (thousands) of binding site energies in one experiment. Using lac repressor as an example, we show that this method has excellent reproducibility giving energy measurements generally consistent within about 0.1kT. While not measuring in parallel as many different sequences as some of the higher throughput methods, we obtain high accuracies because we measure exactly what is necessary, the relative affinity to a large collection of sequences, without any mathematical fitting procedures or approximations required. It is similar to MITOMI in the number of different sites that can be analyzed in parallel, but it is much simpler to perform, requiring only a means to separate bound and unbound fractions which are then sequenced using standard high throughput, short read sequencing machines that are now readily available. When applied to the lac repressor we obtain, in a single experiment, data covering a large fraction of all the previous studies, plus thousands of additional variants, allowing us to compile a much more comprehensive profile of its specificity. We learn that the lac repressor can bind to sites of different lengths but that the preferred sequence, and the mode of binding, depends on the length. We also apply the method to two other members of the LacI/GalR protein family, PurR and YcjW, to obtain extensive models of their specificity and test the generality of the lac repressor's ability to bind to operators of variable length. We find that the lac repressor is apparently unique in its ability to bind with high affinity to sites of different lengths and with different modes of binding.
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Project description:High-Resolution DNA Binding Specificity Analysis of Yeast Transcription Factors
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Project description:Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.
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Project description:We present PIGEON-FEATHER, a method for calculating free energies of opening (∆Gop) at single- or near-single-amino acid resolution for protein ensembles of all sizes from hydrogen exchange/mass spectrometry (HX/MS) data. PIGEON-FEATHER disambiguates and reconstructs all experimentally measured isotopic mass envelopes using a Bayesian Monte Carlo sampling approach. We applied PIGEON-FEATHER to reveal how E. coli and human dihydrofolate reductase (ecDHFR, hDHFR) have evolved distinct conformational ensembles tuned to their catalytic cycles. We showed how two competitive inhibitors of ecDHFR arrest its ensemble in different ways, only one of which is compatible with vertebrate DHFR orthologs. Finally, we uncovered how the lac repressor (LacI) ensemble responds to binding its inducer molecule and operator DNA to regulate transcriptional activation.
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Project description:Transcriptome comparison of the Streptococcus pneumoniae strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose, revealed the elevated expression of various genes and operons, including the lac gene cluster that is organized into two operons i.e. lac operon-I (lacABCD) and lac operon-II (lacTFEG). Deletion of the DeoR family transcriptional regulator lacR that is present downstream of the lac gene cluster, revealed elevated expression of lac operon-I, even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of lac operon-I that encodes enzymes involved in the Tagatose-6-P pathway in the absence of lactose or galactose. Deletion of lacR did not affect the expression of lac operon-II that encodes for a lactose-specific PTS. This finding was further confirmed by ?-galactosidase assays with PlacA-lacZ and PlacT-lacZ in the presence of either lactose or glucose as a sole carbon source in the medium. This suggests the presence of another transcriptional regulator in the regulation of lac operon-II, which could be the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of lac operon-II in the presence of lactose and CcpA-independent regulation of the lac gene cluster in S. pneumoniae. This SuperSeries is composed of the SubSeries listed below.
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