Turnip Yellow Mosaic Virus RNA-Dependent RNA Polymerase: Initiation of Minus Strand Synthesisin Vitro

Abstract

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3′-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3′-tRNA-like structure (TLS). Minus strands were shown to arise byde novoinitiation with the insertion of GTP opposite the penultimate (C) residue of the 3′-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AlMV) RNA, which does not contain a 3′-TLS, was a very poor template. However, other viral RNAs with a 3′-TLS andin vitrotranscripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AlMV 3′-RNA, even when provided with a 3′-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that 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.