Archaeal histone stability, DNA binding, and transcription inhibition above 90 degrees C.
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 4h at 95 degrees C in the presence of 1M KCl. Continued incubation of rHMfA and rHMfB at 95 degrees C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95 degrees C when the salt environment was reduced to 200 mM K Cl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95 degrees 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 degrees C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90 degrees 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.
Soares
D
D
Department of Microbiology, Ohio State University, Columbus 43210, USA.
Dahlke
I
I
Li
W T
WT
Sandman
K
K
Hethke
C
C
Thomm
M
M
Reeve
J N
JN
D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D014158:Transcription, Genetic
D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D006657:Histones; D011994:Recombinant Proteins