MINIMAL TEMPLATE REQUIREMENTS FOR INITIATION OF MINUS-STRAND SYNTHESIS IN-VITRO BY THE RNA-DEPENDENT RNA-POLYMERASE OF TURNIP YELLOW MOSAIC-VIRUS

From mutational analysis of the 3'-terminal hairpin of turnip yellow m osaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TY MV RNA for initiation of minus-strand synthesis by the viral RNA-depen dent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drasticall y, and deletion of only the 3'-terminal A residue results in a fivefol d drop in efficiency. The two C residues of the 3' ACCA end are requir ed for efficient transcription, as shown by substitution mutations. Ho wever, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the ha irpin stem and loop upstream of the 3' ACCA end also do not interact w ith the RdRp in a base-specific way. However, for efficient transcript ion, the hairpin stem should be at least five bp in length, while the calculated Delta G value should be less than -10.5 kcal/mol. Unexpecte dly, the use of nonstructured C-rich RNA templates showed that the RdR p can start internally on an NCCN or NUCN sequence. Therefore, a possi ble function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.