Mechanism of promoter repression by Lac repressor-DNA loops

Abstract

The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.

Figure 1.

Hypothetical contributions to local bidentate repressor concentration at a bacterial operator (O) include contributions because of free repressor and contributions because of DNA-bound repressor at auxiliary operators (O_aux) through DNA looping.

Figure 2.

Promoter–reporter constructs to compare repression by upstream (A and B) versus downstream (C and D) DNA loops in the presence (A and C) or absence (B and D) of IPTG inducer. The dashed region indicates the RNA polymerase footprint, emphasizing that repressor binding at O₂ occludes promoter access.

Figure 3.

RR behavior of the indicated promoter–reporter constructs. UV5 promoter elements (−35, −10), center-to-center spacing of weak and strong lac operators (O₂ and O_sym, respectively), Shine-Dalgarno (sd) and reporter (lacZ) are shown, as well as the length of any insert between the transcription start site (broken arrow) and proximal O₂ operator. Boxed data show RR values for the indicated operator combinations, where filled black rectangles indicate intact operators and open slashed rectangles indicate disrupted operators. RR values in bold highlight comparisons. (A and B) Upstream loops with operators in phase (A) or out of phase (B). (C and D) Downstream loops with operators in phase (C) or out of phase (D).

Figure 4.

Repression data and fits to thermodynamic model for loops with upstream (A and B) versus downstream (C–F) auxiliary O_sym operators. Reporter activity is shown as E' (A, C and E) where the shaded envelope in (A) indicates behavior of upstream loop constructs under induced (open symbols, dashed fit curve) or repressed (filled symbols, solid fit curve) conditions with fits to thermodynamic model. Blue symbols for spacings closer than the vertical line at 41 bp (C–F) indicate constructs that do not show a canonical looping pattern. Panels (B, D and F) show RR data and fits to the thermodynamic model (see Supplementary Figure S3). Filled triangles and dashed fits indicate RR data for strains containing only an auxiliary O_sym operator in the absence of a proximal operator. Grey fits in panels (D) and (F) indicate RR behaviour of upstream loops from (B). Green symbols and fits in panels (E) and (F) show data obtained in the absence of functional Lac repressor (LacI Y282D). Data and fit in magenta (panel F) show the modified RR for downstream loop constructs where the numerator reflects reporter expression in the absence of functional repressor (LacI Y282D) and the denominator reflects repressed reporter expression in the presence of wild-type Lac repressor.

Figure 5.

Models for possible in vivo behavior of upstream (A) or downstream (B) looping constructs bearing a lac UV5 promoter (black dots indicate −35 and −10 elements) for RNA polymerase (red circle) where the proximal lac O₂ operator impinges on the promoter. Possible outcomes under repressing conditions include polymerase exclusion (C and F), polymerase trapping (D and G) or polymerase read-through (E and H).

Figure 6.

RNA polymerase and σ⁷⁰occupancy of lac promoters as detected by ChIP and quantitative PCR. Upstream loop constructs are studied under induced (A) or repressed (B) conditions. Downstream loop constructs are studied under induced (C) or repressed (D) conditions. Black dots indicate −35 and −10 elements. PCR primer sites are indicated by single-headed arrows. ChIP results under conditions of repression (grey bars) or induction (black bars) are shown for α subunit of RNA polymerase (E) or σ⁷⁰ protein (F).

Figure 7.

Models for possible in vivo behavior of upstream (A) or downstream (B) looping constructs bearing a T7 RNA polymerase (red triangle) promoter (oval) that does not overlap with lac O₁ or O_sym operators (rectangles). The promoter DNA is curved by looping in (A) but not (B). Possible outcomes under repressing conditions include polymerase exclusion (C and F), polymerase trapping (D and G) or polymerase read-through (E and H).

Figure 8.

In vivo reporter gene expression from constructs bearing a T7 RNA polymerase promoter (P) two helical turns of DNA upstream of O₁ (BL1093) and with an additional O_sym either further upstream (BL1076) or further downstream (BL1095). Promoter/operator configurations are summarized later, and details are provided in Supplementary Figure S4. In each case, the RR corresponds to the ratio of the height of the final bar to that of the penultimate bar.