JOURNAL OF VIROLOGY, July 1995, p. 4020-4028 Vol. 69, No. 7

Synthesis of Novel Products In Vitro by an RNA-Dependent RNA Polymerase

Chuanzheng Song and Anne E. Simon

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, Amherst, Massachusetts 01003

Received 12 December 1994/Accepted 20 March 1995

RNA-dependent RNA polymerase from turnip crinkle virus-infected turnip transcribes both strands of a virus-associated satellite RNA, sat-RNA C (356 bases), in vitro. While both plus- and minus-strand sat-RNA C can direct the synthesis of full-length complementary-strand products, transcription of minus-strand RNA also generates two non-template-sized products, L-RNA and S-RNA (C. Song and A. E. Simon, Proc. Natl. Acad. Sci. USA 91:8792-8796, 1994). Here we report that synthesis of L-RNA and S-RNA results from terminal elongation of the 3' end of the template. L-RNA has a panhandle structure and is composed of minus-strand template covalently linked to newly synthesized RNA complementary to its 5' 190 bases. S-RNA is composed of template covalently linked to its full-length complementary strand. All minus-strand templates tested yielded S-RNA. However, synthesis of L-RNA was affected by deletion of the 3' end of the minus-strand template or several internal regions and base alterations near the 5' end or in an internal sequence immediately upstream from the template-product junction that could potentially form a heteroduplex with the 3' end. Furthermore, mutations that disrupted or restored a stem-loop involved in RNA recombination in viva affected the level of L-RNA produced in vitro, suggesting that the mechanisms for intramolecular formation of panhandle RNAs and intermolecular RNA recombination involve similar features.