In vitro transcription by the turnip yellow mosaic virus RNA polymerase: acomparison with the alfalfa mosaic virus and brome mosaic virus replicases

Recently, we showed that the main determinant, in the tRNA-like structure o f turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitr o is the 3' ACCA end. By mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in th at it has the lowest Delta G value and a high transcription efficiency, Wit h a nested set of RNA fragments, we show that the minimum template length i s 9 nucleotides and that transcription is improved with increasing the leng th of the template. The results also suggest that proper base stacking cont ributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. Howeve r; the position of the internal initiation site in the template is importan t, and competition between the different sites takes place. Internal initia tion was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AIMV), The BMV polymerase can start internally on ACCA sequences, though inefficiently, Unexpectedly, the polym erases of both AIMV and BMCT can start efficiently on an internal AUGC sequ ence.