Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments

Abstract

E. coli RNA polymerase holoenzyme forms a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay. Here, the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of phage T7 DNA which contains no promoters or TB (tight binding; heparin-resistant) sites is reported. Under the ionic conditions investigated [50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl)piperazine-N''-ethanesulfonic acid (pH 7.7)], both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter reaction. When the DNA fragment is self-ligated into a circular molecule (non-supercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced. This directly proves previous hypotheses that holoenzyme recognizes and preferentially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments. The residual mode of binding of holoenzyme detected with the circular DNA was considered in determining the amount of protein bound at ends only. To calculate end-binding constants (KE), the amount of protein bound nonspecifically (which does not appear to cause efficient filter retention) was also taken into consideration. At 0.degree. C, KE was (2.1 .+-. 0.5) .times. 108 M-1, in good agreement with that determined earlier. This value of KE is relatively constant over the temperature range 0.degree.-37.degree. C. The magnitude of KE indicates that ends can effectively compete with some promoters for RNA polymerase. Therefore, for in vitro promoter binding studies where enzyme is not in excess, end binding (like nonspecific binding) must be considered in the analysis of the promoter binding data, as discussed earlier. The apparent greater specificity for DNA ends of holoenzyme relative to core polymerase is discussed in terms of a steric model in which the .sigma. subunit helps to reduce the affinity of holoenzyme for interior DNA sites through unfavorable steric contacts that are absent in an end complex.