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RNA (1997), 3:1401-1412.
RNA
promoters located on (-)-strands of a subviral RNA associated with
turnip crinkle virus
HANCHENG GUAN, CHUANZHENG SONG, and ANNE E. SIMON
Department of Biochemistry and Molecular Biology and Program in
Molecular and Cellular Biology, University of Massachusetts, Amherst,
Massachusetts 01003
Satellite (sat-) RNA C, one of the nonessential subviral RNAs of
turnip crinkle virus (TCV), is dependent on the TCVencoded
RNA-dependent RNA polymerase (RdRp) for its replication. Earlier work
showed that a stem-loop structure at the 3' end of (+)-strand sat-RNA
C is required for synthesis of (-)-strands in vitro using a partially
purified, template-specific TCV RdRp (Song C, Simon AE, 1995, J Mol
Biol 254:6-14). Cis-sequences on (-)-strands of sat-RNA C that can
serve as separate promoters in vitro have now been defined. Two
promoter sequences are located on (-)-strand sat-RNA C, one comprising
11 bases located near the 3' end, and the other consisting of 14 bases
located 41 bases from the 5' end. Both promoter sequences contain
multiple consecutive C residues followed by multiple consecutive
purines and have no obvious secondary structure, suggesting that,
along with hairpin structures, specific primary sequences can be
recognized by the TCV RdRp. The 3'-proximal promoter sequence directed
synthesis from the 3' terminus using (-)-strand templates with the
natural sat-RNA 3' end (AUCCC-3'). When plasmid-derived bases were
present at the 3' ends of the templates, both promoter sequences could
direct the RdRp to initiate transcription internally at the multiple
consecutive C residues within the promoters. This result suggests that
multiple consecutive C residues are important for transcription
initiation and that natural 3'-end sequences, when located at 3'
termini, help the RdRp to initiate at the 3' end of the molecule.
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