FIG. 3.
Effects of ribonucleotides (A) and pyridine nucleotides (B) on NadR DNA binding. Panels present band shift results obtained by using His-tagged NadR and radiolabelled nadA operator DNA fragments (225-bp PCR product from pFP132). Basic assay conditions were as described in Materials and Methods, with 6.5 nM NadR being used in each reaction mixture. Panel A shows the effect of ribonucleotides on NAD-independent DNA binding by NadR. NAD and various ribonucleotides were added as indicated. Panel B shows the identity of the NadR corepressor. Basic assay conditions were the same as for panel A except that 60 μM ATP was added to all binding reaction mixtures. The indicated pyridine nucleotide cycle intermediates (nucleotide) were all added at 660 μM. QA, quinolinic acid; NA, nicotinic acid; NAMN, nicotinic acid mononucleotide; NAm, nicotinamide; NAAD, nicotinic adenine dinucleotide.