A modified bacterial one-hybrid system yields improved quantitative models of transcription factor specificity
AuthorsChristensen, Ryan G.
Schriefer, Lawrence A.
Wolfe, Scot A.
Stormo, Gary D.
UMass Chan AffiliationsDepartment of Biochemistry and Molecular Pharmacology
Program in Gene Function and Expression
Document TypeJournal Article
Two-Hybrid System Techniques
High-Throughput Screening Assays
Genetics and Genomics
MetadataShow full item record
AbstractWe examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis-GRaMS (Growth Rate Modeling of Specificity)-that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well-characterized C(2)H(2) zinc-finger TF on both a 28 bp randomized library for the standard B1H method and on 6 bp randomized library for the CV-B1H method for which 45 different experimental conditions were tested: five time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media.
SourceNucleic Acids Res. 2011 Jul 1;39(12):e83. Epub 2011 Apr 20. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/43942
Related ResourcesLink to Article in PubMed
Rights© The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.