In this study, a protein designated as HMt (Histone Methanobacterium thermoautotrophicum), was isolated and characterized from Methanobacterium thermoautotrophicum H strain. HMt preparations contain two polypeptides designated as HMtA and HMtB encoded by the genes hmtA and hmtB. These genes were cloned, sequenced and expressed in Escherichia coli. The HMtA and HMtB are predicted to contain 68 and 67 amino acid residues, and have calculated molecular masses of 7,270 and 7,141 Da respectively. Archaean histones form two groups based on N-terminal residues. Archaean A-histones has an alanine or glycine residue at position 2. Archaean B-histones retain the N-terminal methionine residues. HMtA histone has alanine residue at position 2 and has two subunits HMT A 1 and HMTA2 which were encoded by the genes hmtAl and hmtA2. Both polypeptides contain amino acid residues and have similar molecular masses. The HMt histones are very small basic proteins and rich in lysine and arginine residues. The net positive charge on HMtAl, HMtA2, and HMtB were found to be +3, + 1 and + 1, respectively while isoelectric points of HMtA 1, HMtA2, and HMtB 10.2,9.2 and 9.2 respectively. The alignment of primary sequences ofHMt with other archaean histones and consensus sequences of the structured central regions of the eukaryal histones revealed, that the archaean histones contain 66-69 amino acids. HMtA and HMtB are 60-90% identical to pairwise alignment with archaean histones. Nineteen positions contain same residues and 17 additional positions contain only 1 of 2 alternative residues in all of the archaean histones. The sequences and locations of the T AT A box promoter elements and ribosome binding sites are very similar upstream of the hmtA and hmtB genes in Methanobacterium thermoautotrophicum and the upstream of the other archaean histones from hyperthermophiles Methanothermus fervidus (HMf), Methanopyrus (HPy) and mesophile Methanobacterium formicicum (HFo).
Computer algorithms predicted secondary structures and revealed - 70% a-helix content, arranged as three a-helices separated by two short p-sheet charged regions which are also supported by CD spectra. Based on the alignment of amino acid sequences with the eukaryal histones, H2A, H2B, H3 and H4, these archaean proteins share a common secondary structure compared with erythrocyte H2A.
The HMt binding compacted linear pUC19 DNA molecules in vitro and therefore increased their electrophoretic mobilities through agarose gels. The HMt-DNA binding compact DNA molecules into nucleosome-like structures apparently identical to those formed by the HMf proteins which were confirmed by electron microscopy. The DNA-binding and compacting activities are also very resistant to heat inactivation when incubated at 950 C for 5 h. This feature is found consistently in proteins from hyperthermophiles. The expression of HMtA and HMtB histones resulted in the synthesis of rHMtA and rHMtB in Escherichia co/i. The rHMtA and rHMtB were also purified and had similar DNA-binding and compacting activities. The HMtA and HMtB exist as homodimers and heterodimers in solution. The recombinant HMtA and HMtB proteins were also resistant to heat.
Determination of DNA sequences ofHMtA and HMtB revealed that 35-40% of amino acid sequences were conserved in the consensus sequence derived for eukaryal histones H2A, H2B, H3 and H4. These archaean polypeptides and eukaryal histones appear therefore to have evolved from a common ancestor and are likely to have related structures and functions. These might be homologous in evolutionary terms and functions, to the structure at the centre of eukaryal nucleosome formed by the histone (H3-H4)2'
The HMt and HMf consist of histone fold with two non-fold residues at both ends and no labile termini. On the basis of high degree of homology with eukaryal histones, the HMt and HMf appeared as true histones and had common ancestry of the archaean histone and histone fold regions.
The absence of histones and the TAT A-box system of transcription initiation in bacteria could be explained by the bacterial and Archaean lineages having diverged before the coevolution of these systems, but this argument does not explain the lack of histones in Crenarchaeota