Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Plasmid. 2013 Jan;69(1):72-80. doi: 10.1016/j.plasmid.2012.09.002. Epub 2012 Sep 16.
Eleven proteins of the Agrobacterium tumefaciens virB operon are required for type IV secretion. All octopine Ti-plasmid pTiA6NC VirB proteins, except VirB8, could be expressed from a cloned monocistronic gene. Accumulation of VirB8 required translation of the upstream virB7 gene. Analysis of chimeric virB8 genes and a newly constructed virB7 deletion mutant Agrobacterium AD1275 showed that translation of virB7, and not the gene product, is required for VirB8 accumulation. Agrobacterium AD1275 accumulated VirB8 and other downstream virB gene products, and could be complemented with only virB7 in trans. In monocistronic virB8, sequences upstream of the virB8 ORF negatively controls virB8 expression possibly through the formation of a secondary structure that occludes both the ribosome binding site and translation start codon. Disruption of the structure through translation of the upstream gene ensures efficient translation of the virB8 mRNA in wild type bacteria. The pTiA6NC virB8 contains two potential translation start sites within the first eight codons. We show that the first AUG is used for virB8 translation initiation. The seven N-terminal residues resulting from translation initiation at the first AUG are required for both tumor formation and stabilization of VirB3. VirB8 and VirB4 are sufficient for the stabilization of VirB3, and VirB7 stabilizes VirB3 indirectly through its effect on virB8 expression.
根癌农杆菌 virB 操纵子的 11 种蛋白是 IV 型分泌所必需的。所有的胭脂碱 Ti 质粒 pTiA6NC VirB 蛋白，除了 VirB8，都可以从克隆的单顺反子基因中表达。VirB8 的积累需要翻译上游的 virB7 基因。嵌合 virB8 基因和新构建的 virB7 缺失突变体 Agrobacterium AD1275 的分析表明，VirB7 的翻译，而不是基因产物，是 VirB8 积累所必需的。Agrobacterium AD1275 积累了 VirB8 和其他下游 virB 基因产物，并且仅通过反式 virB7 就可以互补。在单顺反子 virB8 中，virB8 ORF 上游的序列负调控 virB8 的表达，可能是通过形成一种二级结构来阻止核糖体结合位点和翻译起始密码子。通过翻译上游基因破坏这种结构，确保了野生型细菌中 virB8 mRNA 的有效翻译。pTiA6NC virB8 在头 8 个密码子内包含两个潜在的翻译起始位点。我们表明，第一个 AUG 用于 virB8 翻译起始。第一个 AUG 翻译起始产生的七个 N 端残基既需要肿瘤形成，也需要 VirB3 的稳定。VirB8 和 VirB4 足以稳定 VirB3，而 VirB7 通过其对 virB8 表达的影响间接稳定 VirB3。
