Translational repression by the bacteriophage T4 gene 32 protein involves specific recognition of an RNA pseudoknot structure.
Translational repression by the bacteriophage T4 gene 32 protein involves specific recognition of an RNA pseudoknot structure.
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DOI：
10.1006/jmbi.1993.1372
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发表时间：
1993-07
影响因子：
5.6
通讯作者：
Yousif Shamoo;Amy Tam;W. Konigsberg;Kenneth R. Williams
中科院分区：
文献类型：
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作者：
Yousif Shamoo;Amy Tam;W. Konigsberg;Kenneth R. Williams
噬菌体T4基因32mRNA5‘端有一个假结，它是由单链核酸结合蛋白基因产物32(Gp32)对基因32(G32)mRNA的自动调节所必需的。结构特异性RNAase的结构图谱表明，由67-碱基配对-52-55(茎-1)和-62-56碱基配对-40-46(茎-2)组成的两个茎区可以折叠成类似于半连续a-螺旋假结的“假结”结构。我们的结果表明，在观察到gp32特异性自动调节的条件下，G32mRNA假结可以形成。虽然G32mRNA假结被镁离子稳定，但它以3‘-发夹结构平衡存在。用限定长度的寡核苷酸进行的凝胶迁移率研究表明，事实上，gp32确实与假结紧密结合。这些研究与McPheeters等人的观点一致，即伪结代表了自体gp32翻译调控所必需的成核位点。尽管这个假结中三级结构的相互作用的中断(与EDTA)显著降低了gp32特异性识别其自身mRNA的能力，但体外诱变研究表明，茎-2和环区的序列(核苷酸-47至-50)也是特异性gp32自我调节的重要决定因素。根据依赖凝胶迁移率变化的竞争分析，伪结的主要元件的重要性顺序是茎-2的茎-1和茎-2或环-2和茎-2。在本实验中，STEM-1的破坏使所得到的结构竞争gp32结合的能力降低了大约四倍。在体外转录/翻译系统中，STEM-1和STEM-2似乎对于维持gp32 mRNA的高水平表达是必不可少的。综上所述，这些结果支持翻译控制模型，在该模型中，伪结区是协同gp32结合的成核点，然后通过包括基因32的起始密码子的长单链RNA在3‘方向进行。
An RNA pseudoknot has been shown to form the 5'-end of bacteriophage T4 gene 32 mRNA that is essential to autoregulation of gene 32 (g32) mRNA by gene product 32 (gp32), a single-stranded nucleic acid binding protein. Structure-mapping of RNA oligonucleotides with structure-specific RNases indicate that two stem regions consisting of nucleotides -67 to -64 base-paired to -52 to -55 (stem-1) and nucleotides -62 to -56 base-paired to -40 to -46 (stem-2) can fold into a "pseudoknotted" structure that may be analogous to the semi-continuous a-helical pseudoknot. Our results suggest that the g32 mRNA pseudoknot can form under conditions where specific autoregulation by gp32 is observed. Although the g32 mRNA pseudoknot is stabilized by Mg2+, it exists in equilibrium with a 3'-hairpin structure. Gel mobility studies carried out with defined length oligonucleotides indicate the gp32 does, in fact, bind tightly to the pseudoknot. These studies agree with the proposal of McPheeters et al., that the pseudoknot represents a nucleation site essential for autogenous gp32 translation regulation. Although disruption of tertiary structure interactions in this pseudoknot (with EDTA) significantly reduces the ability of gp32 to specifically recognize its own mRNA, in vitro mutagenesis studies suggest the sequence of stem-2 and of the loop region (nucleotides -47 to -50) also represent important determinants for specific gp32 autoregulation. Based on a competition assay relying on gel mobility shifts, the order of importance of the major elements of the pseudoknot are stem-1 > sequence of stem-2 or loop-2 > stem-2. In this assay, disruption of stem-1 decreased the ability of the resulting structure to compete for gp32 binding by approximately fourfold. Both stem-1 and stem-2 appear to be essential to maintain high-level expression from gp32 mRNA in an in vitro transcription/translation system. Taken together, these results support the translation control model in which the pseudoknot region is a nucleation point for cooperative gp32 binding, which then proceeds in a 3' direction through a long stretch of single-stranded RNA that includes the initiation codon for gene 32.