The lac repressor is a DNA-binding protein that inhibits the expression of genescoding for proteins involved in the metabolism of lactose in bacteria. These genes are repressed when lactose is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is converted into allolactose, which inhibits the lac repressor's DNA binding ability, thereby increasing gene expression.
Function
The lac repressor operates by a helix-turn-helix motif in its DNA-binding domain, binding base-specifically to the major groove of the operator region of the lac operon, with base contacts also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. This bound repressor can reduce transcription of the Lac proteins by occluding the RNA polymerasebinding site or by prompting DNA looping. When lactose is present, allolactose binds to the lac repressor, causing an allosteric change in its shape. In its changed state, the lac repressor is unable to bind tightly to its cognate operator. Thus, the gene is mostly off in the absence of inducer and mostly on in the presence of inducer, although the degree of gene expression depends on the number of repressors in the cell and on the repressor's DNA-binding affinity. Isopropyl β-D-1-thiogalactopyranoside is a commonly used allolactose mimic which can be used to induce transcription of genes being regulated by lac repressor.
Structure
Structurally, the lacrepressor protein is a homotetramer. More precisely, the tetramer contains two DNA-binding subunits composed of two monomers each. Each monomer consists of four distinct regions:
An N-terminal DNA-binding domain
A regulatory domain
A linker that connects the DNA-binding domain with the core domain
A C-terminal tetramerization region
DNA binding occurs via an N-terminal helix-turn-helixstructural motif and is targeted to one of several operator DNA sequences. The O1operator sequence slightly overlaps with the promoter, which increases the affinity of RNA polymerase for the promoter sequence such that it cannot enter elongation and remains in abortive initiation. Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by a single tetramer induces DNA looping.
Search kinetics of DNA binding
It is hypothesized that lacI and other transcriptions factor's find their binding sites by facilitated diffusion, a combination of free diffusion in 3D and 1D-sliding on the DNA. During sliding, the repressor is in constant contact with the DNA helix, sliding around it and thus facilitating the search process by reducing dimensionality. After probing on average 45 bp by sliding, the TF detaches spontaneously and resumes exploring the genome in 3D. During sliding, LacI is believed to slide over the O1 operator several times before it binds which suggests a trade-off between fast searching on nonspecific and binding to specific sequences. Sliding of the lac repressor on DNA is impossible to observe using fluorescent microscopy, but an all-atom molecular dynamics simulation suggests that the transcription factor encounters a barrier of 1 kBT for sliding and 12 kBT for dissociation, implying that the repressor will slide over 8 bp on average before dissociating. The in vivo search model for the lac repressor includes intersegment transfer and hopping as well as crowding by other proteins which make the genome in E.coli cells less accessible for the repressor.
