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Mechanism of Promoter Repression by Lac Repressor–DNA Loops Nicole A 156–166 Nucleic Acids Research, 2013, Vol. 41, No. 1 Published online 9 November 2012 doi:10.1093/nar/gks1011 Mechanism of promoter repression by Lac repressor–DNA loops Nicole A. Becker1, Justin P. Peters1, Troy A. Lionberger2 and L. James Maher III1,* 1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905, USA and 2Howard Hughes Medical Institute and Jason L. Choy Laboratory of Single-Molecule Biophysics, Department of Physics, University of California, Berkeley, CA 94720, USA Received June 29, 2012; Revised October 1, 2012; Accepted October 2, 2012 ABSTRACT presence of allolactose or its analog, isopropyl b-D-1- thiogalactopyranoside (IPTG), relieving repression. In the The Escherichia coli lactose (lac) operon encodes absence of glucose, RNA polymerase binds cooperatively the first genetic switch to be discovered, and lac with catabolite activator protein at the lac promoter (positive remains a paradigm for studying negative and control). In simplest terms, the mechanism of negative positive control of gene expression. Negative control involves Lac repressor binding to occlude access control is believed to involve competition of RNA of RNA polymerase holoenzyme to the lac promoter (4). polymerase and Lac repressor for overlapping Of particular significance to the present work is the binding sites. Contributions to the local Lac repres- fascinating observation that two remote auxiliary oper- sor concentration come from free repressor and re- ators (Oaux) exist in the lac operon (5). It has been pressor delivered to the operator from remote proposed and demonstrated (6–13) that bidentate repres- auxiliary operators by DNA looping. Long-standing sor tetramers bound at auxiliary operators increase the effective repressor concentration at the regulatory questions persist concerning the actual role of DNA operator (O) through DNA looping (Figure 1). This looping in the mechanism of promoter repression. concept has been exploited as an approach to probe the Here, we use experiments in living bacteria to physical properties of bacterial DNA in vitro (14) and resolve four of these questions. We show that the within the bacterial nucleoid in vivo (12,15–17). distance dependence of repression enhancement is The natural auxiliary operators of the lac operon have comparable for upstream and downstream auxiliary different affinities and occur such that one is just upstream operators, confirming the hypothesis that repressor and one is far downstream of the regulatory operator im- concentration increase is the principal mechanism mediately proximal to the promoter. We have designed of repression loops. We find that as few as four experiments in living bacteria to clarify the role of DNA turns of DNA can be constrained in a stable loop looping in repression. We first tested the hypothesis that by Lac repressor. We show that RNA polymerase increasing the local concentration of repressor at the proximal operator is necessary and sufficient to explain is not trapped at repressed promoters. Finally, we promoter repression (13). This hypothesis implies that show that constraining a promoter in a tight DNA identical auxiliary operators positioned equivalent dis- loop is sufficient for repression even when tances either upstream or downstream of the regulatory promoter and operator do not overlap. operator should provide comparable enhancement of repression by equally increasing the effective local repres- sor concentration at the regulatory operator. Artificial lac INTRODUCTION operators have been shown to enhance repression when It is difficult to overstate the historical significance of the upstream or downstream of the promoter (18), but the Escherichia coli lactose (lac) operon as a paradigm for equivalence of upstream and downstream auxiliary negative and positive control of gene expression (1). This operators has not been previously tested in systematic ex- short segment of the bacterial chromosome encodes a periments. Second, we determined the smallest possible genetic switch that senses and responds to glucose and DNA loop involving Lac repressor in living bacteria. lactose concentrations to produce lactose digestion enzymes Third, we tested the early proposal that favorable only when glucose is absent and lactose is present (2). repressor-polymerase contacts trap RNA polymerase at Central to this function is the homotetrameric Lac repressor repressed promoters (19,20). Fourth, we tested in vivo a protein that binds DNA operator sequences in the lac recent hypothesis that promoter DNA bending strain is operon (3). Lac repressor binding is weakened in the intrinsically repressive (21). *To whom correspondence should be addressed. Tel: +1 507 284 9041/98; Fax: +1 507 284 2053; Email: [email protected] ß The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]. Nucleic Acids Research, 2013, Vol. 41, No. 1 157 0 TCA3CAG and LJM-4111 5 -CTGT3GATG2TG2T2A2 CG2CG3ATGTCACATGAGCTGTCT2CG2TATCGTC, with the mutation underlined. LacI Y282D mutant looping constructs were placed on the single-copy F128 episome by homologous recombination. After mating and selection, correct strain recombinants were confirmed by PCR amplification to detect the inactivated internal 0 Figure 1. Hypothetical contributions to local bidentate repressor con- lacZ O2 sequence with PCR primers LJM-1930 (5 -CGT 0 centration at a bacterial operator (O) include contributions because of CGT4ACA2CGTCG) and LJM-1931 (5 -CAT2GA3GT2 free repressor and contributions because of DNA-bound repressor at A2TGA2TAGCAC). The LacI Y282D mutation was auxiliary operators (Oaux) through DNA looping. confirmed by PCR amplification followed by PCR 0 product sequencing using primers LJM-4112 (5 -A2G2C GACTG AGTGC ATG) and LJM-4113 (50-GA C TGT MATERIALS AND METHODS 2 2 3 2 CGTGC2AGCTG). All data are included in Sup- Bacterial strains plementary Table S2. For studies of T7 RNA polymerase promoters, promoter–reporter constructs were created FW102 (the kind gift from F. Whipple) is a streptomycin based on plasmid pJ992, with modifications illustrated in resistant derivative of CSH142 [araD(gpt-lac) ] and is 5 Supplementary Figure S4, and were placed on the single- designated as wild-type in this study (22). Gene deletions copy F128 episome. Cells were transformed with a and the presence of looping assay episomes were con- plasmid expressing an arabinose inducible T7 RNA poly- firmed by diagnostic polymerase chain reaction (PCR) merase gene (a kind gift from Troy A. Lionberger) before amplification after conjugation and selection (23). lac reporter gene analysis. To determine b-galactosidase activity, bacterial cultures were grown in MOPS minimal DNA constructs buffered media (Teknova) supplemented with 0.8% of DNA looping constructs were based on plasmid pJ992, glycerol, 1.32 mM of dibasic potassium phosphate, created by modifications of pFW11-null (22) as previously 10 mM of NaHCO3, 0.2% of casamino acids and described (23). The relationship between lac operator 12.5 mg/ml of thiamine. Cultures were grown either in sequence and repression in the absence of DNA looping the presence or absence of 2 mM IPTG and/or 0.02% ara- were measured in preliminary experiments (Supplemen- binose. b-Galactosidase assays were performed as tary Figure S1). Sequences of new experimental and described (23). T7 RNA polymerase promoter–reporter control promoters are shown in Supplementary Figure constructs are illustrated schematically in Supplementary S2 with descriptions in Supplementary Table S1. The O2 Figure S4, with data shown in Supplementary Table S3. operator normally present within the lacZ coding region was destroyed by site-directed mutagenesis (15). The Chromatin immunoprecipitation experimental strong UV5 promoter does not contain a Escherichia coli cultures were grown to log phase in 50 ml catabolite activator protein binding site. lacZ looping con- cultures of LB medium at 37C in the presence or absence structs were placed on the single copy F128 episome by of 2 mM IPTG. Cross-linking of DNA and protein homologous recombination between the constructed complexes was accomplished with the addition of 37% plasmids and bacterial episome followed by mating. formaldehyde (Sigma) to make a final concentration of F128 carries the lacI gene producing wild-type levels of 1% in the presence of 10 mM sodium phosphate (pH repressor. Bacterial conjugation and selections were as 7.6). Cultures were maintained at room temperature previously described (23). with constant gentle swirling for 20 min. Reactions were quenched with cold 2 M glycine (200 mM of final concen- In vivo DNA looping assay and data fitting tration). Cells were harvested by centrifugation, washed three times with 4 ml of cold phosphate buffered saline Analysis of lac reporter gene expression was performed as and were resuspended in 1 ml of IP buffer [100 mM of described (23). Raw b-galactosidase reporter activity (E)is Tris–HCl pH 8.0, 300 mM of NaCl, 2% of Triton E0 presented in Miller units. Normalized values are then X-100, 1 mM of PMSF and an additional protease inhibi- obtained by dividing E values by E obtained for a test tor mix (Roche)]. Cells
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