Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.
Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.
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DOI：
10.1002/j.1460-2075.1990.tb07446.x
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发表时间：
1990-08
期刊：
影响因子：
11.4
通讯作者：
Lamb, R A
中科院分区：
文献类型：
--
作者：
Horvath, C M;Williams, M A;Lamb, R A
先前对B型流感病毒RNA片段7的核苷酸序列分析表明，除了编码248个氨基酸的M1蛋白的阅读框外，还有一个585个核苷酸的第二重叠阅读框（BM 2 ORF），其具有195个氨基酸的编码能力。为了寻找BM 2 ORF的多肽产物，在大肠杆菌中表达了基因工程的β-半乳糖苷酶-BM 2 ORF融合蛋白，并制备了纯化的融合蛋白的多克隆兔抗血清。该抗血清用于鉴定在流感B病毒感染的细胞中合成的多肽，命名为BM 2蛋白（Mr约等于12，000）。为了理解从流感B病毒RNA片段7产生BM 2蛋白的机制，对克隆的DNA进行突变分析，并在真核细胞中表达改变的DNA。来自改变的DNA的M1和BM 2蛋白的表达模式表明，BM 2蛋白的起始密码子与M1蛋白的终止密码子在重叠的翻译终止-起始五核苷酸TAATG中重叠，并且BM 2蛋白的表达需要M1合成的5 ′-邻近终止。我们的数据表明，终止-再起始方案是用于翻译的双顺反子mRNA来自流感B病毒RNA片段7，这种策略有一些类似于原核耦合停止-启动翻译串联顺反子。
Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.