Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

High resolution specificity of DNA sequencing highlights alternative mode of lac repressor binding

ABSTRACT: 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.

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Gary Stormo

PROVIDER: E-GEOD-61223 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:In this work, we used our recently developed method Spec-seq to characterize the binding specificity of Glucocorticoid receptor in vitro. This GR Spec-seq experiment has been run twice separately (Dec 2014 and Mar 2015) with different library compositions. The basic workflow is the same as our previous work for lac repressor published in Genetics 198.3 (2014): 1329-1343. Recombinant human GR protein was used to facilitate in vitro DNA-binding and separation experiments. Bound and Unbound DNA fragments were separated in EMSA gels, purified, barcoded for further Illumina sequencing. An initial experiment was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Bound/unbound fractions resulting from EMSA of these libraries and conditions were used to generate PWMs as described. The GR PWM that was generated through this analysis was used to define relative binding energies using the patser program (35), which can be accessed online at (http://stormo.wustl.edu/consensus/cgi-bin/Server/Interface/patser.cgi ). Derived binding affinities are proportional to the inverse of the natural log of the calculated energies.

Dataset Information

High resolution specificity of DNA sequencing highlights alternative mode of lac repressor binding

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure