Archaeal histone stability, DNA binding, and transcription inhibition above 90°C

Abstract

The DNA binding and compacting activities of the recombinant (r) archaeal histones rHMfA and rHMfB from Methanothermus fervidus, and rHPyA1 from Pyrococcus species GB-3a, synthesized in Escherichia coli, have been shown to be completely resistant to incubation for 4 h at 95°C in the presence of 1 M KCl. Continued incubation of rHMfA and rHMfB at 95°C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95°C when the salt environment was reduced to 200 mM KCl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95°C in 1 M KCl, and reducing the salt concentration did not affect the heat resistance of rHPyA1. rHPyA1–DNA complexes remained intact at 100°C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90°C. Transcription in vitro from the P. furiosus gdh promoter was reduced by rHPyA1 binding, in a manner that was dependent on the histone-to-DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to rHPyA1 binding than transcription from a linear template, consistent with rHPyA1 binding introducing physical barriers to transcription and causing changes in the topology of circular templates that also reduced transcription.