Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
PLoS One. 2011 Feb 8;6(2):e16822. doi: 10.1371/journal.pone.0016822.
Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAAUG and ∼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAAUG, known as the 'termination upstream ribosome binding site' (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAAUG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.
终止依赖性重新起始用于协调调节二联体流感 B 段 7 RNA 的 M1 和 BM2 开放阅读框（ORF）的表达。BM2 ORF 的起始密码子与 M1 ORF 的终止密码子重叠在五核苷酸 UAAUG 中，约 10%的核糖体在 M1 终止密码子处终止后重新起始翻译重叠的 AUG。BM2 合成需要存在并通过 UAAUG 上游的 45 个核苷酸的 RNA 进行翻译，该区域称为“终止上游核糖体结合位点”（TURBS）。该区域可能在终止后将核糖体 40S 亚基固定在 mRNA 上，并且 TURBS 的短区域，基序 1，与 18S rRNA 的螺旋 26 互补，已被牵连在这个过程中。在这里，我们使用针对反义寡核苷酸的靶向和酵母细胞中的功能分析提供了 mRNA 和 rRNA 之间直接相互作用的进一步证据。TURBS 还结合起始因子 eIF3，我们在这里表明，当添加外源性时，该蛋白刺激野生型和缺陷型 TURBS 的重新起始，也许通过稳定核糖体-mRNA 相互作用。此外，我们表明 TURBS 相对于 UAAUG 重叠的位置是关键的，并且 18S 互补序列下游的终止会抑制该过程，可能是由于 40S 固定减少所致。然而，在重新起始密码子单独移动到下游的报告 mRNA 中，终止-重新起始被抑制但未被消除，因此重新起始的位置有些灵活。在 eIF4G 耗尽的 RRL 中，远距离 AUG 的重新起始不受抑制，这表明固定的 40S 亚基可以在没有线性扫描要求的情况下移动一定距离。
