Fig 1.
B204 activity and nucleic acid affinity. The release of phosphate (Pi) by B204 during nucleotide hydrolysis was measured using a malachite green assay. (a) Influence of pH on the activity of B204 ATP hydrolysis at 80°C. (b) Effect of temperature on the activity of B204 ATP hydrolysis at pH 4.5. (c) Nucleotides hydrolyzed by B204 at pH 4.5 and 80°C. (d) Divalent metal cations used by B204 in ATP hydrolysis at pH 4.5 and 80°C. (e) Electrophoretic mobility shift assay of DNA in complex with B204. B204 binds linear, supercoiled, and relaxed dsDNA and circular ssDNA at pH 4.5 (22°C). The size of the linear dsDNA molecule is 1.8 kbp. (f) B204 in complex with short dsDNA oligonucleotides at pH 5.0 (22°C). M, Fermentas Ultra-Low range DNA size marker. In panels e and f, the numbers above the wells indicate μg of B204/100 ng DNA. An S with an arrow indicates the nucleic acid shift upon binding to B204. (g) Effect of nucleic acids on the activity of B204 ATP hydrolysis at pH 4.5 80°C. (h) B204 RNA affinity. Electrophoretic mobility shift assay of RNA molecules in complex with B204. B204 binds linear ssRNA (a 700-nt-long fragment prepared from plasmid pEM15 (57) and dsRNA (bacteriophage φ6 genomic RNA) at pH 4.5, 22°C. R, ssRNA size marker (RiboRuler RNA ladder, HighRange; Fermentas); D, dsDNA size marker (GeneRuler 1kb; Fermentas).