Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase

Partially purified RNA-dependent RNA polymerase (RdRp) isolated from plants infected with turnip crinkle virus (TCV) is capable of template-dependent synthesis of TCV-associated RNAs. To determine the cis-sequences required for the synthesis of TCV satellite (sat-) RNA C (-) strands in vitro, templates containing interior deletions were subjected to transcription using RdRp-active fractions. Results indicated that the promoter for (-)-strand synthesis was contained within the 3'-terminal 29 bases of the (+)-strand. Structural probing by enzymatic digestion and chemical modification revealed the presence of a hairpin structure within this terminal region. Compensatory exchanges of four bases in the lower stem or alterations in the sequence and size of the loop region did not affect in vitro transcription, implying that the primary sequence in the loop and lower part of the stem is not important for interaction with the viral RdRp. However, single mutations in the base of the stem or double mutations in the upper stem strongly reduced template activity in vitro, suggesting that the stability of the hairpin is an important functional consideration. Relocation of the 3'-terminal 37 bases containing this stem-loop to inactive template RNA rendered the resultant hybrid RNA competent for in vitro transcription by RdRp activity, suggesting that the promoter for (-)-strand synthesis in vitro is completely contained within the 3'-terminal region.