Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Abstract

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA 1-5 . It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the μs timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves ~50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model 1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.*These authors contributed equally to this work. †Corresponding authors.

The N-terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition

“…The DNA-binding specificity of transcription factors (TFs) is key to gene regulation and thus cellular function. − Regulation of this DNA binding is the end point of many signal-transduction pathways, linking extracellular stimuli to gene-expression responses. − The molecular details of protein–DNA recognition and selectivity are thus of great biochemical interest and biological significance, as are the mechanisms by which DNA-binding proteins can rapidly identify their specific DNA binding sites from among a multitude of nonspecific DNA sites . There has been substantial experimental , and computational − progress toward understanding how transcription factors search DNA for their target sites. This has been aided by recent improvements in computational power that enable the study of protein–DNA recognition on the microsecond time scale using all-atom models …”

Single-Molecule Tracking Approaches to Protein Synthesis Kinetics in Living Cells

“…The technique further requires a microscope setup with high enough temporal and spatial resolution, as well as single photon detection. Recently, a combination of the MINFLUX tracking principle with correlation spectroscopy and a polarization-sensitive detection scheme was used to experimentally resolve the rotational sliding of a DNA-binding protein along the DNA, for the first time . The study was performed on flow-stretched DNA in vitro, but with the combination of, e.g., electroporation, the same principle should be applicable for detecting changes in binding state also in live cells.…”

“…Recently, a combination of the MINFLUX tracking principle with correlation spectroscopy and a polarization-sensitive detection scheme was used to experimentally resolve the rotational sliding of a DNA-binding protein along the DNA, for the first time. 86 The study was performed on flow-stretched DNA in vitro, but with the combination of, e.g., electroporation, the same principle should be applicable for detecting changes in binding state also in live cells. In this case, the improved time resolution, down to a sub millisecond range, would provide completely new possibilities for binding kinetics measurements in the living cell.…”

The N-Terminal Helix-Turn-Helix Motif of Transcription Factors MarA and Rob Drives DNA Recognition

“…It is well-known from structural studies that MarA inserts both of its HTH motifs into the major grooves of the DNA duplex (referred to as the A-and B-boxes in this context, see Figure 1), thus bending the DNA upwards by 35°1 9 . In contrast, Rob only inserts its Nterminal HTH motif into the A-box of the DNA duplex, while the C-terminal HTH lies on the surface of an unbent DNA duplex (Figure 1) 23 .…”

“…The molecular details of protein-DNA recognition and selectivity are thus of great biochemical interest and biological significance, as are the mechanisms by which DNA-binding proteins can rapidly identify their specific DNA binding sites from amongst a multitude of nonspecific DNA sites 8 . There has been substantial experimental 9,10 and computational [11][12][13] progress towards understanding how transcription factors search DNA for their target sites. This has been aided by recent improvements in computational power that enable the study of protein-DNA recognition on the microsecond timescale using all-atom models 11 .…”