Structure/Function Relations of Ribosomal Proteins

核糖体蛋白的结构/功能关系

• 批准号: 6432051
• 负责人: DENNIS A TORCHIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432051
• 关键词: RNA binding protein; binding sites; genetic translation; messenger RNA; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; ribosomal proteins

Binding of the protein S4 to 16S rRNA is critical for the subsequent binding of other ribosomal proteins, and regulates its own translation and that of three other ribosomal proteins. We solved the solution structure of its mRNA binding domain binding domain (S4delta41, 159 residues). S4delta41 has a novel fold with a positively charged cleft running along two distinct domains, suggesting a likely RNA-binding site. We examined the binding of S4delta41 to 112 nucleotides from the mRNA, by mapping changes chemical shifts onto the residues in the protein. Although the residues with perturbed shifts were on the positively charged face of the protein, broadening of certain peaks suggests that the protein-RNA complex is dynamic. Moreover, band shift assays showed that nonspecific binding occurs. We have probed pH, buffer, salt concentration, and temperature, but have not yet identified conditions which abolish non-specific binding. We have shown that the structure of the C-terminal 158 residues of the intact protein is the same that of S4delta41, while the 45 N-terminal residues are very flexible. Chemical shifts and NOESY restraints indicated that residues, S12RRL15 and P30YPP33, adopt transiently ordered structures. In addition, 15N relaxation data indicated that their motion is more restricted than that of other residues in the N-terminus. A BLAST search revealed that R13, R14, P33, G34, and H36 are completely conserved in S4 molecules from both eubacterial and chloroplast ribosomes. The modeled structures indicated that the two conserved arginine side chains of S12RRL15 extend out into solvent in a parallel fashion, suggesting a possible RNA-binding site, and that P30YPP33 form a nascent turn of a polyproline II helix, a protein-protein interaction motif. L11 is a large subunit protein that is essential for efficient protein synthesis. We previously determined the solution structure of it C-terminal domain and have now obtained spectra of intact L11. After finding conditions that maximize solubility we will determine its structure. L11 has not be located in the 2.4? crystal structure of large subunit of the ribosome. The difficulty of placing L11 may reflect L11's proposed role as a molecular switch. NMR is well-suited to determine if the linker between the N- and C-terminal domains is flexible, thereby allowing L11 to act as a molecular switch

蛋白S4与16S rRNA的结合对于其他核糖体蛋白的随后结合至关重要，并调节其自身的翻译以及其他三种核糖体蛋白的翻译。 我们解决了其mRNA结合结构域结合结构域的溶液结构（S4DELTA41，159个残基）。 S4DELTA41具有新颖的折叠，沿两个不同的域沿着带正电荷的裂缝呈正，这表明可能是RNA结合位点。我们通过映射化学位移到蛋白质中的残基来研究了从mRNA的S4DELTA41与112个核苷酸的结合。尽管具有扰动偏移的残基在蛋白质的正电荷面上，但某些峰的扩展表明蛋白-RNA复合物是动态的。此外，频带移位测定表明发生非特异性结合。 我们已经探测了pH，缓冲液，盐浓度和温度，但尚未确定废除非特异性结合的条件。我们已经表明，完整蛋白的C末端158残基的结构与S4DELTA41的结构相同，而45个N末端残基非常灵活。化学位移和噪声限制表明，残基S12RRL15和P30YPP33采用瞬时有序结构。 此外，15N松弛数据表明，其运动比N末端中的其他残基更受限制。爆炸搜索表明，R13，R14，P33，G34和H36在叶绿体和叶绿体核糖体的S4分子中完全保守。 模型的结构表明，S12RRL15的两个保守精氨酸侧链以平行方式延伸至溶剂，表明可能的RNA结合位点，P30YPP33形成了多发性II螺旋的新生转弯，一种蛋白质蛋白质相互作用基序。 L11是一种大型亚基蛋白，对于有效的蛋白质合成至关重要。我们先前确定了IT C末端结构域的溶液结构，并已获得完整的L11光谱。在发现最大化溶解度的条件之后，我们将确定其结构。 L11不在2.4中？核糖体大亚基的晶体结构。 放置L11的困难可能反映出L11作为分子开关的作用。 NMR非常适合确定N-和C末端域之间的接头是否灵活，从而使L11充当分子开关