Proteins mediating DNA loops effectively block transcription

Abstract

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop‐mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

Protein-mediated looping of DNA under tension requires supercoiling

“…All DNA fragments for TPM experiments were amplicons of PCR reactions with plasmid DNA pYY_I1_400_BstEII ( 37 ) or pO1O2 ( 26 )) as templates with dNTPs (Fermentas-Thermo Fisher Scientific Inc., Pittsburgh, PA, USA), digoxigenin- and biotin-labeled primers (Integrated DNA Technologies, Coralville, IA, or Invitrogen, Life Technologies, Grand Island, NY, USA) ( Supplementary Table S1 ) and Taq Polymerase (New England BioLabs, Ipswich, MA, USA). The total lengths of the DNA constructs Os-400-O1 and O1-400-O2 were 909, and 831 bp respectively with centrally located, 400 bp, LacI-inducible loops as diagramed in Supplementary Figure S1 .…”

Protein-mediated loops in supercoiled DNA create large topological domains

“…Finally, non-specific binding of LacI to DNA is negligible (if not non-existent) in the buffer condition and LacI concentration used, as seen by atomic force imaging ( 26 , 27 ) and tethered particle microscopy assays where, in the absence of supercoiling, the extension of a DNA tether without operator sequences in the presence of LacI remained constant in time ( 28–31 ).…”

Studying protein-DNA interactions using atomic force microscopy

“…The accuracy of these measurements depends on uniformity of lengths of the DNA population. Modifications, such as biotinylation [8] or looping [9], to a specific end of a DNA substrate can help determine its orientation.…”