Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy

摘要: The influence of heparin and heparan sulphate (HepS) on the appearance analysis open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) linear DNA using ex situ imaging atomic force microscopy (AFM) has been investigated. Introducing or HepS into reaction mix significantly reduces non-specific interactions σ70RNAP RNAP after RPo allowing for better interpretation complexes shown within AFM images, particularly templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors σRNAP binding formation, would also inhibit template to mica support surface thereby lower yield active RNAP-DNA complexes. We found reverse this true, detected AFM, a simple tandem gene model two λPR promoters, increased. Moreover unexpectedly, efficient heparin, with both them having dispersive effect sample, minimising unwanted RNAP-RNAP well between template. success method relied observation highest affinity all molecular components. For our system, three constituent biopolymers muscovite RNAP>Heparin HepS>DNA. While we observed can mica, presence overcomes greater RPos analysis. This be extended other proteins enzymes, which have an higher DNA, improve sample preparation studies.