Structure/function Relations Of Ribosomal Proteins

核糖体蛋白的结构/功能关系

• 批准号: 6664129
• 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/6664129
• 关键词: RNA binding protein; binding sites; messenger RNA; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure function; ribosomal proteins; translation factor; RNA binding protein; binding sites; messenger RNA; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure function; ribosomal proteins; translation factor

Binding of protein S4 to 16S rRNA is critical for the subsequent binding of other ribosomal proteins. In addition S4 regulates its own translation and that of three other ribosomal proteins. Following our description of the solution structure of S4's mRNA binding domain (S4delta41, 159 residues), we showed that the structure of the C-terminal 158 residues of the intact protein is the same that of S4delta41. In contrast, the 45 N-terminal residues are highly flexible, but contain two conserved regions, S12RRL15 and P30YPP33, that adopt transiently ordered structures in solution. These transiently ordered conformations, predicted by NMR, were similar to those observed in the independently determined crystal structure of the 30S ribosomal unit from T. thermophilus. During the course of the structural work on the S4 N-terminus, severe overlap problems were encountered in the NMR spectra resulting from chemical shift degeneracy and the high concentration of proline residues. To overcome these problems, we developed two novel experiments that used the carbonyl chemical shifts to remove signal degeneracy. These experiments enabled us to make complete signal assignments of the N-terminal domain of intact S4. 15N relaxation data (T1, T2, and {H}15N NOE) have been collected on both S4 and S4delta41,. These data were analyzed using an anisotropic Model-free approach. The analysis yielded average overall correlation times and anisotropies of rotational diffusion of 8.39 plus/minus .02 ns and 1.53 plus/minus .04 for S4delta41, and 11.40 plus/minus .10 ns and 1.41 plus/minus .06 for S4. In addition transverse relaxation dispersion measurement indicated that there were no motions on the millesecond-to-microsecond timescale within the C-terminal 159 residues of the protein. Taken together, the relaxation data indicate the C-terminal 159 residues of S4 reorient as an axially symmetric rigid body. This observation suggests that this portion of S4 and acts as a folding template upon which ribosomal RNA can organize.

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。另外，S4还调节其自身的翻译和其他三种核糖体蛋白的翻译。按照我们描述了S4 mRNA结合结构域的溶液结构（S4DELTA41，159个残基），我们表明，完整蛋白的C末端158残基的结构与S4DELTA41的结构相同。相比之下，45个N末端残基具有高度柔韧性，但包含两个保守区域S12RRL15和P30YPP33，它们采用溶液中瞬时有序结构。 NMR预测的这些瞬时构象与在嗜热链球菌的30S核糖体单位的独立确定的晶体结构中观察到的构象相似。 在S4 N末端的结构工作过程中，在化学移位退化和脯氨酸残基的高浓度导致的NMR光谱中遇到了严重的重叠问题。为了克服这些问题，我们开发了两个新的实验，这些实验使用了羰基化学位移来消除信号脱位。这些实验使我们能够对完整S4的N末端域进行完整的信号分配。 在S4和S4DELTA41上都收集了15N放松数据（T1，T2和{H} 15N NOE）。使用各向异性模型方法分析了这些数据。该分析得出的平均总相关时间和旋转扩散的各向异性为8.39 plus/sinus 0.02 ns和1.53 plus/sinus .04，S4DELTA41，11.40 plus/sinus .10 ns和1.41 plus/sig/sirus .06。另外，横向松弛分散度测量表明，在蛋白质的C末端159残基内，毫秒到微秒的时间尺度上没有运动。综上所述，弛豫数据表明S4重新定位的C末端159残基作为轴向对称的刚体。该观察结果表明，S4的这一部分是核糖体RNA可以组织的折叠模板。