TURNIP YELLOW MOSAIC-VIRUS RNA-DEPENDENT RNA-POLYMERASE - INITIATION OF MINUS-STRAND SYNTHESIS IN-VITRO

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubili zed from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, microc occal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3'-fragments as short as a 28-nucleotide-long RNA co mprising the amino acid acceptor stem of the 3'-tRNA-like structure (T LS). Minus strands were shown to arise by de novo initiation with the insertion of GTP opposite the penultimate (C) residue of the 3'-termin al -CCA The TYMV RdRp activity was template specific in that poly(A) R NA was not copied, and alfalfa mosaic virus (AIMV) RNA, which does not contain a 3'-TLS, was a very poor template. However, other viral RNAs with a 3'-TLS and in vitro transcripts of tRNAs were copied to varyin g degrees. Fully modified tRNAs were either inactive or poorly active templates, and AIMV 3'-RNA, even when provided with a 3'-terminal -ACC A, was not copied detectably. A potential role of the acceptor stem ps eudoknot as a promoter element was assessed with mutations that drasti cally altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show t hat RNAs with both a tRNA-like conformation and a -CCA 3'-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot. (C) 1997 Academic Pre ss.