Enhancement of LacI binding in vivo

Abstract

Transcription factors (TFs) bind to specific sequences in DNA to regulate transcription. Despite extensive measurements of TFs’ dissociation constant (Kd) in vitro, their apparent Kdin vivo are usually unknown. LacI, a bacterial TF, is often used to artificially recruit proteins onto eukaryotic genomes. As LacI binds tightly to its recognition site (LacO) in vitro with a Kd about 10 picomolar (pM), it is often assumed that LacI also has high affinity to LacO in vivo. In this work, we measured LacI binding in living yeast cells using a fluorescent repressor operator system and found an apparent Kd of ∼0.6 μM, four orders of magnitude higher than that in vitro. By genetically altering (i) GFP-LacI structure, (ii) GFP-LacI stability, (iii) chromosome accessibility and (iv) LacO sequence, we reduced the apparent Kd to <10 nM. It turns out that the GFP tagging location and the fusion protein stability have a large effect on LacI binding, but surprisingly, chromosome accessibility only plays a mild role. These findings contribute to our quantitative understanding of the features that affect the apparent Kd of TF in cells. They also provide guidance for future design of more specific chromosomal recruitment through high-affinity TFs.

Tuning up Transcription Factors for Therapy

“…An even higher discrepancy was found between the in vivo and in vitro data in yeast, where LacI-GFP fusions bound the DNA with micromolar dissociation rate constants [82]. This weak binding was shown to be sensitive to the nature of protein fusions [82], which possibly explains, why more careful design of the activation domain linkage is needed for LacI fusions.…”

“…Above the critical point, unintended consequences can occur. Thus it can be useful to keep the intracellular concentration of the TF at intermediate or low level, which can be achieved by increasing reducing the translation by stem-loops, by increasing the degradation rate of the protein and by using appropriate promoters [52,82]. An increase of the degradation rate affects the TF bound to the DNA, reducing its local effect; therefore, using a weaker promoter to drive the expression of the TF may be the preferable option [82].…”

Tuning up Transcription Factors for Therapy

“…An even higher discrepancy was found between the in vivo and in vitro data in yeast, where LacI-GFP fusions bound the DNA with micromolar dissociation rate constants [90]. This weak binding was shown to be sensitive to the nature of protein fusions [90], which possibly explains why a more careful design of the activation domain linkage is needed for LacI fusions.…”

“…Above the critical point, unintended consequences can occur. Thus, it can be advantageous to keep the intracellular concentration of the TF at intermediate or low level, for example, by reducing translation, by increasing the degradation rate of the protein or by using appropriate promoters [61,90]. An increase of the degradation rate affects the TF bound to the DNA, reducing its local effect; therefore, using a weaker promoter to drive the expression of the TF may be the preferred option [90].…”

Chemically Induced Chromosomal Interaction (CICI): A New Tool to Study Chromosome Dynamics and Its Biological Roles

“…For this assay to work, it is essential to keep the concentrations of the LacI-FKBP12 and TetR-FRB fusion proteins at low levels: freely diffusing proteins dimerize with the chromatin-bound counterparts and therefore interfere with the interaction between the two targeted loci. Based on our previous study 29 , we constructed strains containing the REV1 promoter driving LacI and TetR fusion proteins, which allows high occupancy of LacO / TetO arrays with low fusion protein concentrations (Methods).…”