Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ~90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.In all kingdoms of life transcription factors (TFs) regulate gene expression by site-specific binding to chromosomal DNA, preventing or promoting the transcription by RNA polymerase. The lac operon of Escherichia coli, a model system for understanding TF-mediated transcriptional control (1), has been the subject of extensive biochemical (2-4), structural (5) and theoretical (6,7) studies since the seminal work by Jacob and Monod (8). However, the in vivo kinetics of the lac repressor, and all other TFs, has only been studied indirectly by monitoring the regulated gene products. Traditionally, this was done on a population of cells (9), in which unsynchronized gene activity among cells masks the underlying dynamics. Recent experiments on single cells allow investigation of stochastic gene expression (10-15). However, direct observation of TF mediated gene regulation (16) remains difficult, because it often involves only a few copies of TF and their chromosomal binding sites. Here we report on a kinetics study of how fast a lac repressor binds its chromosomal operators and dissociates in response to a metabolic signal in a living E. coli cell.Single molecule detection also makes it possible to investigate how a TF molecule searches for specific binding sites on DNA, a central question in molecular biology. Target location by TFs (and most nucleic acid binding proteins) is believed to be achieved by facilitated diffusion, in which a TF searches for specific binding sites through a combination of one-dimensional (1D) diffusion along a short DNA segment and 3D translocation among DNA segments through cytoplasm (17). However, real-time observation in living cells has not been available because of technical difficulties. Here we report on such an investigation, providing quantitative information of the search process.Correspondence to: X. Sunney Xie. † These authors contributed equally to this work. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2010 April 13. To image the lac repressor, we expressed it from the native chromosomal lacI locus as a Cterminal fusion with the rapidly maturing (~7 min) yellow fluorescent protein (YFP) Venus (A206K) (15,20) (Fig. 1A). The short...

It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple onedimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1 D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 ± 0.2 k B T. Such small barriers facilitate rapid search for binding sites.Many nucleic acid enzymes and proteins that act on DNA quickly locate target sites by diffusing along nonspecific DNA. It has been shown that proteins can both hop and slide along doublestranded DNA [1][2][3] , although the microscopic mechanism of protein motion along DNA molecules is still not understood in molecular detail. In particular, the path traced by a sliding protein molecule along the surface of DNA has not been established. Both linear paths, parallel to the DNA axis, and helical paths, following a strand or groove of the DNA around the DNA axis, have been taken as assumptions in biophysical and biochemical models. Although rotation of sliding proteins around the DNA helix was implicitly 4 and explicitly 5,6 anticipated, such rotation was not shown to occur during diffusive sliding. The concept of rotational coupling has also arisen among structural biologists based on concepts of molecular recognition and observations of detailed structural complementarity between proteins and DNA 7-9 . Despite © 2009 Nature America, Inc. All rights reserved.Correspondence to: Biman Bagchi; X Sunney Xie.Correspondence should be addressed to B.B. (bagchibiman@yahoo.com) the persistent high profile of this question in the literature, it remains unknown whether sliding proteins track the DNA helix. Such tracking would have major biophysical and biochemical implications: for example, only a limited set of enzyme-helix juxtapositions would need to be considered in questions of protein-DNA interaction. In this work, we examine the dependence on protein size of the diffusion constant for sliding along DNA in order to distinguish pure translational diffusion (Fig. 1a) along DNA from rotation-coupled (or -slaved) diffusion (Fig. 1b). The result offers insights into the mechanism of target search and recognition of all DNAbinding proteins.As a protein moves along DNA, it experiences three different frictional forces arising from rando...

Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which—together with molecular dynamics simulations—suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.

The dynamic impact approach (DIA) represents an alternative to overrepresentation analysis (ORA) for functional analysis of time-course experiments or those involving multiple treatments. The DIA can be used to estimate the biological impact of the differentially expressed genes (DEGs) associated with particular biological functions, for example, as represented by the Kyoto encyclopedia of genes and genomes (KEGG) annotations. However, the DIA does not take into account the correlated dependence structure of the KEGG pathway hierarchy. We have developed herein a path analysis model (KEGG-PATH) to subdivide the total effect of each KEGG pathway into the direct effect and indirect effect by taking into account not only each KEGG pathway itself, but also the correlation with its related pathways. In addition, this work also attempts to preliminarily estimate the impact direction of each KEGG pathway by a gradient analysis method from principal component analysis (PCA). As a result, the advantage of the KEGG-PATH model is demonstrated through the functional analysis of the bovine mammary transcriptome during lactation.