Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1

ABSTRACT The minus-strand promoter of Alfalfa mosaic virus (AMV), a tripartite plant virus belonging to the family Bromoviridae, is located within the 3′-terminal 145 nucleotides (nt), which can adopt a tRNA-like structure (TLS). This contrasts with the subgenomic promoter for RNA4 synthesis, which requires ∼40 nt and forms a single triloop hairpin. Detailed analysis of the minus-strand promoter now shows that a similar triloop hairpin, hairpin E (hpE), is crucial for minus-strand synthesis. The loop sequence of hpE appeared to not be essential for RNA synthesis, whereas the identity and base-pairing capability of bases below the triloop were indeed essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site similar to the process of subgenomic transcription. Similar effects were observed when deleting (part of) the TLS, suggesting that tertiary contacts between hpE and the TLS prevent internal initiation. The data indicate that the minus-strand promoter hpE and the subgenomic promoter hairpin are equivalent in binding the viral polymerase. We propose that the major role of the TLS is to enforce the initiation of transcription by polymerase at the very 3′ end of the genome.

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

Journal of Virology ◽

10.1128/jvi.74.1.264-271.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 264-271 ◽

Cited By ~ 22

Author(s):

Birgit A. L. M. Deiman ◽

Paul W. G. Verlaan ◽

Cornelis W. A. Pleij

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Transcription Initiation ◽

Alfalfa Mosaic Virus ◽

Brome Mosaic Virus ◽

Yellow Mosaic Virus ◽

Turnip Yellow Mosaic Virus ◽

Virus Rna ◽

Internal Initiation

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 ΔG 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.

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