Single-molecule studies of repressor–DNA interactions show long-range interactions

Abstract

We have performed single-molecule studies of GFP-LacI repressor proteins bound to bacteriophage DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths.binding coefficient ͉ multiple sites ͉ transcription factors O ne of the great challenges in protein-DNA interaction studies is to obtain information from single protein-DNA molecules, rather than ensembles of millions of molecules. To understand protein-DNA interactions at the single-molecule level, single protein molecules must be imaged with high resolution to resolve two key fundamental issues: the number of bound proteins and their locations, which requires that the DNA be extended in a linear manner. We have developed an integrated photon molecular counting (IPMC) method to determine the number of proteins bound to DNA and a platform incorporating nanofluidic channels and total internal reflection fluorescence (TIRF) microscopy to determine the location of the lactose repressor protein (LacI) bound to DNA carrying multiple tandem lac operator (lacO) insertions.Since its use by Jacob and Monod (1) in Escherichia coli as the fundamental model for gene regulation, the LacI repressor has been one of the most intensively studied proteins in molecular biology (2-4). LacI normally exists as a tetramer consisting of two tightly coupled dimers with a dimer-dimer dissociation constant K d of Ϸ10 Ϫ13 M (5). Because, in principle, tetramers can bind to two remote sites and, thus, form protein-DNA complexes, in this experiment we worked with LacI monomers to avoid aggregation problems. LacI monomers bind strongly to lacO sequences (K d Ϸ 10 Ϫ10 M; ref. 6), and the expected off rate k off is on the order of hours (6). This slow rate allows us ample time for observation of the repressor-DNA complexes. We used a DNA construct containing 256 contiguous copies of the lacO target sequence (7,8) (Fig. 1a). Each binding unit consists of a palindromic 21-bp lacO binding site, followed by a 15-bp nonspecific sequence. Because of this 15-bp spacer, we might expect that at the 60 nM monomer protein concentration used in our experiments, all 256 sites would be occupied by two GFP-LacI monomers (Fig. 1b), and we might expect no steric interaction between the contiguously bound GFPLacI molecules. As described below, this is not the case (Fig. 1c).The key idea of the IPMC method is that a fluorescent molecule will emit a mean number of photons N 0 before it irreversibly bleaches. If the emitted number of photons N t from an illuminated region is integrated until the region is bleached, the total number of molecules tha...

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Transport phenomena in nanofluidics

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