A novel molecular switch

Abstract

Transcriptional regulation is a fundamental process for regulating the flux of all metabolic pathways. For the last several decades, the lac operon has served as a valuable model for studying transcription. More recently, the switch that controls the operon has also been successfully adapted to function in mammalian cells. Here we describe how, using directed evolution, we have created a novel switch that recognizes an asymmetric operator sequence. The new switch has a repressor with altered headpiece domains for operator recognition and a redesigned dimer interface to create a heterodimeric repressor. Quite unexpectedly, the heterodimeric switch functions better than the natural system. It can repress more tightly than the naturally occurring switch of the lac operon; it is less leaky and can be induced more efficiently. Ultimately, these novel repressors could be evolved to recognize eukaryotic promoters and used to regulate gene expression in mammalian systems.

Figure 1. Operator sequences used in reporter plasmids

The operator sequences are aligned and numbered with respect to the central base pair. The top sequence is the natural (0R1) operator of the lac operon. The second sequences represents the ideal symmetric lac operator (212) followed by the third operator which corresponds to the gal-like operator (412). The 411 operator diverges the gal-like operator further at position 6. The final two operators are chimeric operators composed of the 212 and 411 half sites. The numerical identifiers associated with each operator correspond to the identity of the bases found in the right half site of each fully symmetric operator (1/2/3/4 = A/C/G/T respectively)

Figure 2. In vivo characterization of various combinations of repressors and operators

Dark shading = no IPTG, Light shading = 2.5 mM IPTG (a) Both the IAN and TAN mutants are capable of repressing the 411 operator with approximately the same affinity as the wild type switch. (b) The two changes in the operator sequences, 212 and 411, allow for significant divergence of the half sites such that the wild type (YQR) and TAN mutant repressors specifically repress their respective operators.

Figure 3. In vivo repression and induction analysis

Dark shading = no IPTG, Light shading = 2.5 mM IPTG (a) Neither the wild type (YQR) nor the TAN mutant repressors are capable of repressing the chimeric operator in their homodimeric state. Only when the two mutants are co-expressed can repression be achieved. (b) When both repressors contain the wild type dimer interface however, a mixed population of dimeric repressors exists, as evidenced by the repression of each of the three operators analyzed. When the Y282A point mutation is introduced into the wild type repressor gene (Black bars), homodimerization (repression of the 212 operator) and heterodimerization (repression of the Chimeric operator) is lost.

Figure 4. Location of residues mutated in redesign of the dimer interface

(a) Structure of the repressor containing 4 domains (b) Residues L251, R255, D278, C281 and Y282 on the repressor make intergenic interactions with residue Y282 on the dimer interface.

Figure 5. In vivo characterization of identified heterodimers

Repression analysis represents the relative ability of each repressor to associate as either a homo-dimer or hetero-dimer depending on the operator used in analysis. The ability of each mutant to heterodimerize with its complimentary monomer (◊ Y282A, - Y282D, ▲ Y282S, ■ C281S/Y282L) is shown on the X axis. Each mutant was assayed in the presence of its complimentary monomer and the chimeric operator. The repression ratio is defined by the ratio between the fluorescent signal when the repressor is absent divided by the signal when the repressor is present. For each sample, the measured repression ratio was normalized to the repression ratio for the wild type switch (●). In an independent assay, the isolated intergenic suppressor mutants were analyzed for their ability to homodimerize and thus repress transcription from the 411 operator. The relative repression ratios compared to the wild type switch are plotted on the Y-axis.

Figure 6. Characterization of the top functioning heterodimeric repressors

(a) Specificity of operator recognition was determined by measuring the repression ratio (GFP signal in absence of repressor/GFP signal with repressor). The heterodimeric repressors cannot repress either the 212 or 411 operators. (b) Fluorescent signals were measured for the top functioning heterodimeric repressors when GFP was controlled by the chimeric operator. All of the hits repress transcription better than the wild type switch. Two of the hits function better than the wild type switch using the ‘ideal’ operator. (c) Dark shading = no IPTG, Light shading = 2.5 mM IPTG. Unlike the wild type switch using the ‘ideal’ operator, each of the heterodimeric switches are capable of full induction. They therefore represent true ‘ideal’ switches since the increase in repression does not limit inducibility. (d) The increased repression seen for the heterodimeric repressor with the chimeric operator (black bar) is also seen when the heterodimers are altered to contain two wild type DNA binding domains (grey bars) and incubated with the natural OR1 operator. Repression for this construct is increased further when the ‘ideal’ symmetric (212) operator is used. Abbreviations- Het1: Y282A & 4A20, Het 2: Y282S & 4S29, Het 3: Y282S & 4S31, Het4: Y282S & 4S36