Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein
Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein
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DOI：
10.1261/rna.1231008
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发表时间：
2008-11-01
期刊：
影响因子：
4.5
通讯作者：
Brown, T. David K.
中科院分区：
文献类型：
--
作者：
Powell, Michael L.;Napthine, Sawsan;Brown, T. David K.
乙型流感病毒的M1和BM2开放阅读框架（orf）通过终止依赖性重启动过程偶联表达。BM2 ORF的AUG起始密码子与上游M1 ORF的终止密码子在五核苷酸UAAUG中重叠，BM2的合成依赖于M1 ORF的翻译和终止密码子。在这里，我们研究了BM2表达所需的mRNA序列。终止再起始依赖于UAAUG五核苷酸上游的45个核苷酸（nt） RNA，其中包括与18S rRNA螺旋26互补的必要拉伸。因此，与杯状病毒类似，mRNA和rRNA之间的碱基配对可能在M1终止密码子终止后将40S亚基拴在mRNA上发挥作用。与此一致的是，在BM2起始密码子下游超过24nt处重新定位M1终止密码子可抑制BM2的表达。RNA结构探测发现，UAAUG重叠上游的RNA结构不高，但当核糖体遇到M1停止密码子时，核糖体可能立即形成茎环59，其中18S rRNA互补区位于顶端环，靠近螺旋26。突变分析表明，BM2表达的正常起始位点选择要求被暂停，启动密码子上下文的影响很小，非规范启动密码子的有效利用。这表明，启动因子的充分补充并不需要重新启动过程。
Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 59 of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.