Tetramer opening in LacI-mediated DNA looping

Abstract

Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations.

Fig. 1.

Structures of Lacl variants, looped complexes, and chemical cross-linkers. (A) LacI tetramer structure with DNA-binding domain in red, protein core in green and tetramerization domain in blue. Each dimer is shown in color tones of different darkness. (B) Different LacI-DNA loop topoisomers. Arrows indicate the 5′-3′ orientation of operators. (C) TPM setup: unlooped and looped configurations highlight the difference of bead mobility. Schematic (not to scale) drawing of DNA construct indicates the lengths of different segments (interoperator distance reported as center-to-center). (D) The E36C (Left) and Q231C (Right) LacI mutants. Each protein is shown in side view (Upper) and top view (rotated by 90° about the horizontal axis, Lower), with each dimer in a different shade of gray. E36C is shown cross-linked with DPDPB and Q231C with BMOE; the cross-linker is shown in red spacefill representation. The tetramer structure shown is a model of the maximal opening compatible with the chosen cross-linker. (E) Chemical structures of the cross-linkers. The external sulfur atoms in each structure are from the cysteine sulfhydryl groups of the cross-linked protein.

Fig. 2.

Representative TPM recordings (Left) obtained with wild-type LacI, uncross-linked E36C, uncross-linked Q231C, and Q231C cross-linked with BMOE. All traces show 〈R〉 obtained by Gaussian filtering with σ = 4 s. The Right panels show histograms of corresponding recordings on the Left.

Fig. 3.

Looping kinetics. (A) Kinetic scheme with two looped states. [R] indicates the concentration of Lac repressor tetramer. (B) Uncross-linked Q231C: a trace of states (red) superimposed on a trace of 〈R〉 (black) with thresholds indicated by the green lines. (C) Distributions of uncross-linked Q231C state durations (black squares) before a particular transition (indicated by an arrow connecting two states) fitted with mono-exponential functions (red lines). These distributions result from the records of 20 tethers with total measurement time of about 13 h. The short and long tether looped states are arbitrarily numbered as 1 and 2, respectively. The resulting rate constants are: k₁₂ = 0.022 ± 0.003 s⁻¹, k_L1 = 0.023 ± 0.003 s⁻¹, k₂₁ = 0.041 ± 0.002 s⁻¹, and k_L2 = 0.046 ± 0.005 s⁻¹.

Fig. 4.

Operator binding. O₁ binding measured at equilibrium in the absence (filled symbols) or in the presence of IPTG (1 mM, open symbols), for E36C (A–C) and Q231C (D–F) uncross-linked or cross-linked as indicated. In B, C, E and F the dotted line shows the binding curve of uncross-linked protein for reference. In C the orange data show the effect of pretreating E36C-DPDPB with DTT. The values of K_D (expressed in pM) obtained by fitting the data are as follows: 14 ± 2 (E36C); 22 ± 3 (E36C-BM[PEO]₂); 12 ± 1 (E36C-DPDPB); 23 ± 2 (Q231C); 320 ± 50 (Q231C-BM[PEO]₂); 240 ± 30 (Q231C-BMOE). For comparison, K_D for wild-type LacI is 15 ± 4 (30).

Fig. 5.

Aggregate distributions of 〈R〉 for Q231C uncross-linked (A) and cross-linked with BM[PEO]₂ (B), or BMOE (C). Histograms are composed from the records of 16 tethers (15 h) for Q231C, 21 tethers (18 h) for Q231C-BM[PEO]₂ and 23 tethers (19 h) for Q231C-BMOE. To minimize the effects of bead-to-bead variability, time records, irrespective of the number of looping states they exhibit, were chosen with the unlooped state within a narrow (10 nm) interval around the maximum (170 nm) of the distribution of the unlooped states of a larger population of tethers (approximately 100). D and E highlight the changes in the distributions induced by cross-linking with BM[PEO]₂ (D) or BMOE (E). The histogram in gray shows the uncross-linked data (the same as in A) for reference; the red bars show the difference between cross-linked and uncross-linked histograms.