The Effect of HU Protein on Lac-Repressor-Mediated DNA Looping

Abstract

The regulation of transcription includes the formation of DNA loops mediated by proteins that bind to DNA. DNA stiffness and supercoiling influence DNA loop-formation and can be modified by abundant nucleoid associated-proteins in bacteria that bind to DNA. Studies indicate substantial redundancy between different nucleoid proteins in vivo, but deletion of the heat unstable protein (HU) makes the formation of short loops between strong recognition sequences dependent on the sequence of the loop segment. The HU protein has two subunits (α and β), induces negative supercoiling of DNA, and changes the flexibility of DNA upon binding non-specifically. For loops longer than a persistence length, for which DNA stiffness is not limiting, the effect of HU on loop formation is not well understood. In tethered particle motion (TPM) experiments, a titration of the formation of a 900 bp loop between strong recognition sequences, OID and O1, as a function of HU concentration exhibits mild changes across a broad range of concentration (0-900 nM). This was surprising since the overall tether length simultaneously decreased which should have facilitated looping. It suggests that HU has no specific effect on the lac repressor-induced DNA loop, the effect of HU is more prominent on shorter loops, or the strong OID operator, which supports up to 90% looping at a variety of loop lengths across a large range of LacI concentrations, overwhelms the effect of HU. Indeed, this last possibility may be significant, since the formation of a 400 bp loop with wild-type spacing and Lac loop operators, O1 and O2, exhibits a narrower range of looping as a function of LacI concentration with a maximum of 50%.