Fluorescence resonance energy transfer over approximately 130 basepairs in hyperstable lac repressor-DNA loops.
Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA. DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes. Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form. Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart. For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form. From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed. Time-resolved FRET suggests that this complex exists in both closed- and open form populations. Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop. This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry. LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions.
Edelman
Laurence M
LM
Department of Chemistry and Biochemistry, University of Maryland, College Park, College Park, Maryland 20742-2021, USA.
Cheong
Raymond
R
Kahn
Jason D
JD
D001426:Bacterial Proteins; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D004735:Energy Transfer; D029968:Escherichia coli Proteins / Q000737:chemistry*; D019793:Fluorescein; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D056968:Lac Repressors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry*; D012235:Rhodamines; D013194:Staining and Labeling / Q000379:methods
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D046911:Macromolecular Substances; D009841:Oligonucleotides; D012097:Repressor Proteins; D012235:Rhodamines; D004247:DNA; D019793:Fluorescein