Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase.
The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.
Vázquez
A L
AL
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias (CSIC), Universidad de Oviedo, 33006 Oviedo, Spain.
Alonso
J M
JM
Parra
F
F
D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D017250:Caliciviridae Infections / Q000821:virology*; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics*; D017354:Point Mutation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012324:RNA-Dependent RNA Polymerase