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 p lants infected with turnip crinkle virus (TCV) is capable of template- dependent synthesis of TCV-associated RNAs. To determine the cis-seque nces required for the synthesis of TCV satellite (sat-) RNA C (-) stra nds in vitro, templates containing interior deletions were subjected t o transcription using RdRp-active fractions. Results indicated that th e promoter for (-)-strand synthesis was contained within the 3'-termin al 29 bases of the (+)-strand. Structural probing by enzymatic digesti on and chemical modification revealed the presence of a hairpin struct ure 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 primar y sequence in the loop and lower part of the stem is not important for interaction with the viral RdRp. However, single mutations in the bas e of the stem or double mutations in the upper stem strongly reduced t emplate activity in vitro, suggesting that the stability of the hairpi n is an important functional consideration. Relocation of the 3'-termi nal 37 bases containing this stem-loop to an inactive template RNA ren dered 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. (C) 19 95 Academic Press Limited