Mutational analysis of the Potyviridae transcriptional slippage site utilized for expression of the P3N-PIPO and P1N-PISPO proteins.-猫眼课题宝

DOI：

10.1093/nar/gkw441

发表时间：

2016-09-19

期刊：

Nucleic acids research

影响因子：

14.9

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Olspert A;Carr JP;Firth AE

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027478/?report=classic

马铃薯病毒科 (Potyviridae) 包括最大且最重要的 RNA 植物病毒家族。一个重要的重叠 ORF，称为 pipo，位于主要多蛋白 ORF 的内部区域。最近，pipo 的表达被证明依赖于保守 GAAAAAA 序列处的程序性转录滑移，导致在一定比例的病毒转录物中插入额外的 A，将 pipo ORF 与多蛋白 ORF 的 5' 三分之一融合在读码框内。然而，介导滑移的序列特征尚未被表征。使用融合到可以自由突变的不同基因组位置的 pipo 滑动位点区域的重复副本，我们研究了转录滑动的序列要求。我们发现前导 G 并不是严格要求的，但侧翼序列 GC 含量的增加与较高的插入率相关。同聚六聚体最适合主要产生单核苷酸插入。我们还发现，在无插入转录本中，紧邻 GAAAAAA 的 3' 端存在过多的 G 到 A 取代，我们推断这是由于正链合成过程中“来回”形式的滑移造成的。对野生型和反向互补序列的分析表明，滑移优先发生在 Poly(A) 合成过程中，因此主要发生在正链合成过程中。

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.