Role of the 3' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase In vitro.
A template-dependent RNA polymerase has been used to determine the sequence elements in the 3' untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3'-terminal 4 nucleotides of domain D1 indicated the importance of the 3'-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3' untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.
Osman
T A
TA
Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom.
Hemenway
C L
CL
Buck
K W
KW
D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010947:Plants, Toxic; D012343:RNA, Transfer / Q000235:genetics*; D012367:RNA, Viral / Q000502:physiology*; D013698:Templates, Genetic; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000502:physiology*; D014779:Virus Replication
D020413:3' Untranslated Regions; D012367:RNA, Viral; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases