Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteinsReport as inadecuate


Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins


Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins - Download this document for free, or read online. Document in PDF available to download.

Publication Date: 2016-05-16

Journal Title: Nucleic Acids Research

Publisher: Oxford University Press

Volume: 44

Issue: 16

Pages: 7618-7629

Language: English

Type: Article

This Version: VoR

Metadata: Show full item record

Citation: Olspert, A., Carr, J. P., & Firth, A. E. (2016). Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins. Nucleic Acids Research, 44 (16), 7618-7629. https://doi.org/10.1093/nar/gkw441

Description: This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/nar/gkw441

Abstract: The Potyviridae comprise the largest and most important family of RNA plant viruses. An essential overlapping ORF, termed pipo, resides in an internal region of the main polyprotein ORF. Recently, expression of pipo was shown to depend on programmed transcriptional slippage at a conserved GAAAAAA sequence, resulting in the insertion of an extra A into a proportion of viral transcripts, fusing the pipo ORF in frame with the 5 third of the polyprotein ORF. However, the sequence features that mediate slippage have not been characterized. Using a duplicate copy of the pipo slip site region fused into a different genomic location where it can be freely mutated, we investigated the sequence requirements for transcriptional slippage. We find that the leading G is not strictly required, but increased flanking sequence GC content correlates with higher insertion rates. A homopolymeric hexamer is optimal for producing mainly single-nucleotide insertions. We also identify an overabundance of G to A substitutions immediately 3'-adjacent to GAAAAAA in insertion-free transcripts, which we infer to result from a ‘to-fro’ form of slippage during positive-strand synthesis. Analysis of wild-type and reverse complement sequences suggests that slippage occurs preferentially during synthesis of poly(A) and therefore occurs mainly during positive-strand synthesis.

Sponsorship: Wellcome Trust (Grant ID: 106207)

Biotechnology and Biological Sciences Research Council (Grant ID: BB/J015652/1)

Biotechnology and Biological Sciences Research Council (Grant ID: BB/J011762/1)

European Research Council (Grant ID: 646891)

Embargo Lift Date: 2100-01-01

Identifiers:

External DOI: https://doi.org/10.1093/nar/gkw441

This record's URL: https://www.repository.cam.ac.uk/handle/1810/256179



Rights: Attribution 4.0 International

Licence URL: http://creativecommons.org/licenses/by/4.0/





Author: Olspert, AllanCarr, John P.Firth, Andrew E.

Source: https://www.repository.cam.ac.uk/handle/1810/256179



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