Biosynthesis of reovirus-specified polypeptides. Effect of methylation on the efficiency of reovirus genome expression in vitro.
Biosynthesis of reovirus-specified polypeptides. Effect of methylation on the efficiency of reovirus genome expression in vitro.
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呼肠孤病毒特异性多肽的生物合成。
DOI：
10.1016/0042-6822(77)90422-6
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发表时间：
1977
期刊：
影响因子：
3.7
通讯作者：
Charles E. Samuel
中科院分区：
文献类型：
--
作者：
Kenneth H. Levin;Charles E. Samuel
在体外研究了甲基化对呼肠孤病毒基因组RNA转录和呼肠孤病毒信使RNA翻译的影响。结果表明：（1）经胰凝乳蛋白酶处理后的呼肠孤病毒颗粒，在S-腺苷-L-蛋氨酸（SAdoMet）和S-腺苷-L-同型半胱氨酸（SAdoHcy）存在下，均能以相似的速率催化呼肠孤病毒RNA的转录。甲基化的病毒呼肠孤病毒RNA合成的SAdoMet的存在下，具有琼脂糖-尿素-聚丙烯酰胺凝胶电泳模式不可区分的非甲基化的mRNA合成的SAdoHcy的存在下。(2)对于甲基化mRNA，foursadn三类呼肠孤病毒信使物种的SAdoMet介导的甲基化的相对水平是相当的; SAdoHcy：SAdoMet比率为350：1时，SAdoHcy使甲基化降低超过98%。(3)甲基化的呼肠孤病毒mRNA在SAdoMet或SAdoHcy存在下通过小麦无细胞系统以相当的效率翻译。在SAdoMet存在下，未甲基化mRNA的翻译效率约为甲基化mRNA的50%;在SAdoHcy存在下，未甲基化mRNA的翻译效率约为甲基化mRNA的50%。(4)聚丙烯酰胺凝胶电泳显示，尽管甲基化mRNA合成的呼肠孤病毒多肽的数量要多得多，但在体外对未甲基化mRNA合成的σ、μ和呼肠孤病毒特异性多肽与对甲基化mRNA合成的多肽在定性上是不可区分的。(5)通过β-消除化学去除甲基化呼肠孤病毒mRNA的5′-末端7-甲基鸟苷残基，使甲基化mRNA的翻译活性降低约75%;在相同条件下处理，未甲基化mRNA的翻译活性仅略有降低。(6)未甲基化的呼肠孤病毒mRNA没有竞争性抑制甲基化mRNA的翻译，而是出现了协同效应。(7)甲基化的呼肠孤病毒mRNA的翻译对7-甲基鸟苷5′-单磷酸（7 mGMP）的抑制敏感，而非甲基化的呼肠孤病毒mRNA的翻译对7 mGMP不敏感;在1 mM或更高的7 mGMP存在下，浓度相当的甲基化和非甲基化mRNA的翻译活性几乎相同。(8)未甲基化的呼肠孤病毒mRNA与小麦核糖体的结合可通过蔗糖密度离心检测到;然而，甲基化的mRNA的结合比未甲基化的mRNA显著更有效。这些结果表明，甲基化对呼肠孤病毒RNA的体外转录没有显著影响，但小麦系统催化的呼肠孤病毒mRNA的翻译效率被病毒mRNA的甲基化大大提高，尽管甲基化似乎不是翻译的强制要求。
The effect of methylation on the transcription of reovirus genome RNA and the translation of reovirus messenger RNAin vitrowas investigated. The following results were obtained: (1) Purified reovirions converted to cores by chymotrypsin treatment catalyzed the transcription of reovirus RNA at comparable rates in the presence of eitherS-adenosyl-l-methionine (SAdoMet) orS-adenosyl-l-homocysteine (SAdoHcy). Methylated virus reovirus RNA synthesized in the presence of SAdoMet possessed an electrophoretic pattern on agarose-urea-polyacrylamide gels indistinguishable from unmethylated mRNA synthesized in the presence of SAdoHcy. (2) The relative level of SAdoMet-mediated methylation of the foursadn threemclass reovirus message species was comparable for methylated mRNA; SAdoHcy at an SAdoHcy:SAdoMet ratio of 350:1 reduced the methylation by more than 98%. (3) Methylated reovirus mRNA was translated by the wheat cell-free system in the presence of either SAdoMet or SAdoHcy with comparable efficiency. Unmethylated mRNA was translated about 50% as efficiently as methylated mRNA in the presence of SAdoMet; in the presence of SAdoHcy, unmethylated mRNA. (4) Polyacrylamide-gel electrophoresis revealed that the σ, μ, and ψ reovirus-specified polypeptides synthesizedin vitroin responce to unmethylated mRNA were qualitatively indistiniguishable from those synthesized in response to methylated mRNA, although quantitatively the amount of reovirus polypeptides synthesized with methylated mRNA was much greater. (5) Chemical removal of the 5′-terminal 7-methylguanosine residue of methylated reovirus mRNA by β-elimination reduced the translational activity of the methylated mRNA by about 75%; the translational activity of unmethylated mRNA was reduced only slightly by treatment under identical conditions. (6) Unmethylated reovirus mRNA did not competitively inhibit the translation of methylated mRNA but, rather, appeared to have a cooperative effect. (7) The translation of methylated reovirus mRNA was sensitive to inhibition by 7-methylguanosine 5′-monophosphate (7mGMP), whereas the translation of unmethylated reovirus mRNA was insensitive to 7mGMP; the translational activities of comparable concentrations of methylated and unmethylated mRNA were nearly identical in the presence of 1 mMor greater 7mGMP. (8) The binding of unmethylated reovirus mRNA to wheat ribosomes was detectable by sucrose density centrifugation; however, the binding of methylated mRNA was significantly more efficient than unmethylated mRNA. These results indicate that methylation has no significant effect on the transcription of reovirus RNAin vitrobut that the efficiency of translation of reovirus mRNA catalyzed by the wheat system is greatly increased by methylation of the viral mRNA although methylation does not appear to be an obligatory requirement for translation.