Thomm, Michael and Sandman, K. and Frey, G. and Koller, G. and Reeve, J.
Transcription In Vivo and In Vitro of the Histone-Encoding
Gene hmfB from the Hyperthermophilic Archaeon
Journal of bacteriology = JB = J. Bacteriol. 174, pp. 3508-3513.
Immediately upstream of the hmJB gene, in a DNA fragment cloned from Methanothermusfervidus, are two
identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which
conforms precisely to the consensus TATA box element proposed for methanogen promoters. By using this
duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus
thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp
downstream from a TATA box, thus providing strong evidence for the functional conservation of this
transcriptional signal in two phylogenetically very diverse methanogens. Transcription of the hmfB gene in vivo
in M. fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and
sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that
the genome ofM. fervidus contains only one copy of the 73-bp sequence upstream of the hmJB gene. Since the
second copy of the 73-bp sequence, presumably generated artifactually during the original hmJB cloning,
functioned equally well as a promoter in the M. thermolithotrophicus transcription system, all information
needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within
this sequence. The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding
protein in M. fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures. Cell-free
transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1
and 1:1, respectively, whereas the archaeal histonelike protein HTa from Thermoplasma acidophilum inhibited
transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU
from Escherichia coli had no detectable effect on transcription.