The loopometer: a quantitative in vivo assay for DNA-looping proteins

Abstract

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this ‘loopometer’ assay to measure DNA looping for a variety of bacterial and phage proteins.

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli

“…Microtiter plate-based LacZ assays were carried out as previously described. 26 Briefly, cultures were grown in M9MM in 96-well microtiter Quantitative Fluorescent Assays. Bacterial cultures (100 μL) were grown in M9MM in 96-well microtiter plates.…”

“…Microtiter plate-based LacZ assays were carried out as previously described . Briefly, cultures were grown in M9MM in 96-well microtiter plates.…”

Transcription disrupts DNA-scaffolded bacteriophage repressor complexes

“…Remarkably, even LacI bound to O2 significantly impedes transcription when it is part of a loop [25, 26]. Many transcription factors secure loops between distant sites [27, 28], and others may wrap DNA [29]. Here, we extended our study of topological impediments of transcription elongation to include the 186 and the λ CI repressors, two cooperatively binding proteins with packaging and regulatory functions that, respectively, wrap or loop DNA [29, 30].…”

“…Thus, just one LacI can bind two operators simultaneously, causing the looping out of the intervening DNA 25,26 . However, many other repressors and, transcription factors in general, cause looping via the interaction between proteins bound at distant sites 27,28 . In yet a different case of cooperativity, proteins may form structures that wrap DNA.…”

RNA polymerase efficiently transcribes through DNA‐scaffolded, cooperative bacteriophage repressor complexes

“…Remarkably, even LacI bound to O2 significantly impedes transcription when it is part of a loop [28,29]. Many transcription factors cooperatively secure loops between distant sites [30,31], and others may wrap DNA [32] or bridge contiguous and/or non-contiguous helices. For example, the histone-like nucleoid structuring (H-NS) protein binds with high affinity to hundreds of sites in E. coli and may cooperatively spread with lower affinity across AT-rich regions [33].…”