The Study of the Structure and Function of RNA Replicase from RNA Coliphage

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To investigate the relationship between the structure and function of RNA replicase of RNA coliphage, gene for the replicase <BETA> -subunit protein was cloned and the replicase activity was examined in vivo after the amino acids in the protein were changed through the base substitutions of the gene. At the first year, the plasmid carrying the intact replicase <BETA> -subunit protein gene was constructed from the cDNA clone of Q <BETA> or SP, and it was found that each <BETA> -subunit gene was expressed and could function as a normal replicase subunit in the cells. At the last year, using the <BETA> -subunit gene of Q <BETA> , the mutant plasmid in which the Gly residue at a consensus segment (Tyr-Gly-Asp-Asp) seen in the putative RNA-dependent RNA polymerases from various viruses including RNA phages has been changed to Ala, Ser, Pro, Met or Val were constructed and the replicase activity was examined in vivo. The cells carrying a plasmid to which the site-specifically mutagenized replicase gene was inserted lost the replicase activity in vivo but prevented the proliferation of the wild-type Q <BETA> and SP phage, by inhibiting the phage RNA synthesis. However, the substitution of the Gly residue at another site showed much less interfering effects. These results suggest that an introduction of amino acid substitutions in the consensus sequence loses the nucleotide polymerizing activity but still retains the template recognition activity of Q <BETA> replicase.