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 a re required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synt hesis 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 c entral core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3 ' -terminal 4 nucleotid es of domain Dl indicated the importance of the 3 ' -terminal CA sequence f or minus-strand synthesis, with the sequence CCCA or GGCA giving the highes t transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop str uctures in domains Dl, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequenc e 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 upstre am of domain D3 was not required for minus-strand synthesis. Template-RNA p olymerase 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 po lymerase with lower affinity.