Abstract
We have analyzed one of the functional domains of Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta. Deletion mapping analysis of the carboxyl-terminal region of the beta-subunit protein revealed that the terminal 18 amino acid residues (positions 571-588) are dispensable for the replicase reaction. Subsequent deletions up to the Ala-565 residue reduced the RNA polymerizing activity of the replicase in vivo but increased it in vitro. The mutant replicases with enhanced in vitro RNA polymerizing activity were found to have relaxed template specificity for ribosomal RNAs and cellular RNAs as well as Qbeta RNA. Deletions beyond the Ile-564 residue abolished both the RNA polymerizing activity and the binding ability to midivariant (MDV)-poly(+) RNA, a derivative of a natural template for Qbeta replicase, MDV-1 RNA. These results suggest that the carboxyl-terminal part of the beta-subunit participates in RNA recognition of Qbeta replicase.
Highlights
An RNA-dependent RNA polymerase (RNA replicase) of RNA coliphage Q consists of four subunits
When the deletion extended beyond the Val-570 residue of the -subunit, despite the fact that the cells expressing the mutant replicases supported poor growth of the invading Qsus51 phage (Fig. 3A), lysates of the cells carrying pRQ1-DR569, -DS568, -DS567, or -DC566 synthesized much more RNA than did lysate from cells harboring pRQ1
We propose that the carboxyl-terminal part of the -subunit of Q replicase is one of the candidates for functional domains determining the template recognition from the following results
Summary
Phage, and Plasmids—Escherichia coli A/ (sup pro/Fϩ) and BE110 (supϩ rel-1 T2r tonA22 phoA4 Hfr) were used as indicators. Plasmid pUC-MDV-LR carrying the segment corresponding to MDV-poly(ϩ) RNA was described previously (10). Construction of Plasmids Carrying the -Subunit Gene—Plasmids carrying the -subunit gene, of which the 3Ј-terminal end was shortened to various extents, were constructed using the polymerase chain reaction method. Oligonucleotide RQ1-f2 has a nucleotide sequence corresponding to positions 3328 –3351 of the Q cDNA sequence including the cleavage site for the restriction enzyme, XhoI, and was used as a forward primer (Table I). Amplification of the DNA fragments was carried out according to the
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