In Vitro Transcription by the Turnip Yellow Mosaic Virus RNA Polymerase: a Comparison with the Alfalfa Mosaic Virus and Brome Mosaic Virus Replicases

Abstract

ABSTRACT Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro 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 that it has the lowest Δ G value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.