title: Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus.
abstract: We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.
authors.affiliation: Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Yoo
authors.forename: M D
authors.initials: MD
authors.lastname: Parker
authors.forename: G J
authors.initials: GJ
authors.lastname: Cox
authors.forename: L A
authors.initials: LA
authors.lastname: Babiuk
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D003062:Codon; D017938:Coronavirus, Bovine / Q000201:enzymology* / Q000235:genetics*; D003545:Cysteine; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli; D015151:Immunoblotting; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D015032:Zinc / Q000378:metabolism*
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; D003545:Cysteine