Selinger D, Brennwald P, Liao X, Wise J A
Department of Biochemistry, University of Illinois, Urbana-Champaign 61801.
Mol Cell Biol. 1993 Mar;13(3):1353-62. doi: 10.1128/mcb.13.3.1353-1362.1993.
Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.
信号识别颗粒（SRP）是一种核糖核蛋白，由六种多肽和一个RNA分子组成。SRP RNA可分为四个结构域，其中最后一个结构域是最保守的，在粟酒裂殖酵母中，它是有害突变定位的主要位置。哺乳动物SRP54蛋白（SRP54p）与大肠杆菌4.5S RNA结合的能力表明，SRP54p可能直接与该区域相互作用。4.5S RNA是SRP RNA的同源物，仅包含结构域IV。为了确定结构域IV对于裂殖酵母细胞中SRP54p结合是否至关重要，我们使用了一种天然免疫沉淀-RNA测序分析方法，来测试13种突变的SRP RNA在体内与该蛋白结合的能力。G156A突变改变了结构域IV末端螺旋非规范第一个碱基对的5'残基，并导致轻微的条件性生长缺陷，该突变会减少RNA与SRP54p的组装。将凸起区域5'处的两个进化上不变的残基之一突变为G156对生长更有害，并且几乎消除了SRP54p的结合。我们得出结论，核苷酸154至156的保守性可能是它们作为SRP54蛋白的序列特异性识别元件的结果。我们还测试了一系列在结构域IV保守的四核苷酸环和相邻茎中进行核苷酸替换的突变体。虽然四核苷酸环突变对生长有害，但它们对SRP54p结合几乎没有影响。破坏四核苷酸环侧翼碱基对的突变会导致条件性生长缺陷，并显著降低与SRP54p的结合。破坏四核苷酸环相邻短茎中的其他两个碱基对，对SRP54p结合有类似但不太显著的影响。这些数据首次证明，SRP RNA结构域IV的序列特异性接触和结构完整性对于与SRP54p的组装都很重要。
