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Journal of Virology, December 2000, p. 11671-11680, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Role of the 3' tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

T. A. M. Osman,1 C. L. Hemenway,2 and K. W. Buck1,*

Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom,1 and Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 276952

Received 19 April 2000/Accepted 25 September 2000

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.

* Corresponding author. Mailing address: Department of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, United Kingdom. Phone: 44 207 594 5362. Fax: 44 207 584 2056. E-mail: k.buck{at}ic.ac.uk.

Journal of Virology, December 2000, p. 11671-11680, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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