Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus strand synthesis in vitro.
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 by de novo initiation 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 (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 varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AIMV 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.
Singh
R N
RN
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301, USA.
Dreher
T W
TW
D001483:Base Sequence; D001937:Brassica; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D017806:Tymovirus / Q000201:enzymology* / Q000235:genetics
D012367:RNA, Viral; D012343:RNA, Transfer; D012324:RNA-Dependent RNA Polymerase