Proteins mediating different DNA topologies block RNAP elongation with different efficiency

Abstract

AbstractMany DNA-binding proteins induce topological structures such as loops or wraps through binding to two or more sites along the DNA. Such topologies may regulate transcription initiation and may also be roadblocks for elongating RNA polymerase (RNAP). Remarkably, a lac repressor protein bound to a weak binding site (O2) does not obstruct RNAP in vitro but becomes an effective roadblock when securing a loop of 400 bp between two widely separated binding sites. To investigate whether topological structures mediated by proteins bound to closely spaced binding sites and interacting cooperatively also represent roadblocks, we compared the effect of the λ CI and 186 CI repressors on RNAP elongation. Dimers of λ CI can bind to two sets of adjacent sites separated by hundreds of bp and form a DNA loop via the interaction between their C-terminal domains. The 186 CI protein can form a wheel of seven dimers around which specific DNA binding sequences can wrap. Atomic force microscopy (AFM) was used to image transcription elongation complexes of DNA templates that contained binding sites for either the λ or 186 CI repressor. While RNAP elongated past λ CI on unlooped DNA, as well as past 186 CI-wrapped DNA, it did not pass the λ CI-mediated loop. These results may indicate that protein-mediated loops with widely separated binding sites more effectively block transcription than a wrapped topology with multiple, closely spaced binding sites.