Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus.
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.
Yoo
D
D
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Parker
M D
MD
Cox
G J
GJ
Babiuk
L A
LA
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*
D003062:Codon; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; D003545:Cysteine