Isolation and characterization of the genes for RNA polymerase subunits from Haloarcula japonica strain TR-1

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We have cloned and sequenced genes for encoding RNA polymerase subunits, RpoA, RpoB1, RpoB2, RpoC, RpoH from extremely halophilic archaeon Haloarcula japonica strain TR-1. Cloned genes, rpoH, rpoB2, rpoB1, rpoA and rpoC, were one operon in this order. Southern hybridization analysis showed that relationships between restriction pattern and its positions on chromosome, was right. Compared with the operon structure between archaea of completed-genome, conservation in the operon structure was observed. In comparison of halophilic microorganisms, the structure was completely conserved. In compared with archaea of completed-genome including methanogens and thermophiles, the operon structure was basically conserved in the order, but some gene fusions by rpoB1 and ropB2, was observed. Construction of phylogenic trees from RpoA and RpoH among archaea of completed-genome, also showed their evolutionary conservation in primary structure (amino acid sequence). In addition, hexa-histidine tagged-expression plasmids for these genes were constructed to overexpress these proteins. Overexpression studies using these plasmids were carried out in Escherichia coli host strains, and expressed products were affinity purified by Ni-columns. Affinity purified proteins, RpoB1 and RpoB2, were separated on SDS-PAGE gel to ascertain its production and purity. These results together will give us to convenient way not only for in vitro reconstitution system of halophile RNA polymerase, but also insight in archaeal transcription studies.
2006-12-20

Isolation and characterization of the genes for RNA polymerase subunits from Haloarcula japonica strain TR-1

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