Fouts D E, True H L, Celander D W
Department of Microbiology and College of Medicine, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
Nucleic Acids Res. 1997 Nov 15;25(22):4464-73. doi: 10.1093/nar/25.22.4464.
The R17/MS2 coat protein serves as a translational repressor of replicase by binding to a 19 nt RNA hairpin containing the Shine-Dalgarno sequence and the initiation codon of the replicase gene. We have explored the structural features of the RNA operator site that are necessary for efficient translational repression by the R17/MS2 coat protein in vivo . The R17/MS2 coat protein efficiently directs lysogen formation for P22 R17 , a bacteriophage P22 derivative that carries the R17/MS2 RNA operator site within the P22 phage ant mRNA. Phages were constructed that contain fragmented operator sites such that the Shine-Dalgarno sequence and the initiation codon of the affected gene are not located within the RNA hairpin. The wild-type coat protein directs efficient lysogen formation for P22 phages that carry several fragmented RNA operator sites, including one in which the Shine-Dalgarno sequence is positioned 4 nt outside the coat protein binding site. Neither the wild-type R17/MS2 coat protein nor super-repressor mutants induce lysogen formation for a P22 phage encoding an RNA hairpin at a distance of 9 nt from the Shine-Dalgarno sequence, implying that a discrete region of biological repression is defined by the coat protein-RNA hairpin interaction. The assembly of RNA species into capsid structures is not an efficient means whereby the coat protein achieves translational repression of target mRNA transcripts. The R17/MS2 coat protein exerts translational regulation that extends considerably beyond the natural biological RNA operator site structure; however, the coat protein still mediates repression in these constructs by preventing ribosome access to linear sequence determinants of the translational initiation region by the formation of a stable RNA secondary structure. An efficient translational regulatory mechanism in bacteria appears to reside in the ability of proteins to regulate RNA folding states for host cell and phage mRNAs.
R17/MS2外壳蛋白通过与一个包含Shine-Dalgarno序列和复制酶基因起始密码子的19个核苷酸的RNA发夹结构结合，作为复制酶的翻译阻遏物。我们已经探索了RNA操纵位点的结构特征，这些特征对于R17/MS2外壳蛋白在体内进行有效的翻译阻遏是必需的。R17/MS2外壳蛋白有效地指导了P22 R17的溶原形成，P22 R17是一种噬菌体P22衍生物，其在P22噬菌体反义mRNA内携带R17/MS2 RNA操纵位点。构建了含有片段化操纵位点的噬菌体，使得受影响基因的Shine-Dalgarno序列和起始密码子不在RNA发夹结构内。野生型外壳蛋白指导携带几个片段化RNA操纵位点的P22噬菌体有效地形成溶原，包括其中一个Shine-Dalgarno序列位于外壳蛋白结合位点外4个核苷酸处的噬菌体。野生型R17/MS2外壳蛋白和超阻遏突变体都不会诱导一个在距Shine-Dalgarno序列9个核苷酸处编码RNA发夹结构的P22噬菌体形成溶原，这意味着由外壳蛋白-RNA发夹相互作用定义了一个离散的生物阻遏区域。将RNA种类组装成衣壳结构并不是外壳蛋白实现对靶mRNA转录本进行翻译阻遏的有效方式。R17/MS2外壳蛋白发挥的翻译调控作用远远超出了天然生物RNA操纵位点结构；然而，外壳蛋白在这些构建体中仍然通过形成稳定的RNA二级结构来阻止核糖体接近翻译起始区域的线性序列决定因素，从而介导阻遏作用。细菌中一种有效的翻译调控机制似乎在于蛋白质调节宿主细胞和噬菌体mRNA的RNA折叠状态的能力。
