RIBOSOMAL PROTEIN S4 INTERACTIONS WITH MRNA AND RRNA

核糖核酸和蛋白质复合体(RNPs)具有许多重要的功能。 单元格，包括翻译。这些复合体中的蛋白质是如何 识别特定位点，促进RNA折叠，并有助于发挥作用 都是重要的问题。这个项目的目标是探索这些 对具有双重功能的核糖体蛋白S4的疑问：它是一个关键 核糖体中的蛋白质组装、结合和组织约500 小亚基RNA的核苷酸结构域，还结合了一个110 在αmRNA中的核苷酸假结结构，在那里它抑制 通过一种新颖的“陷阱”机制进行翻译。蛋白质是有组织的 至少分为三个区域：N-末端片段(42个残基)不是 核糖体组装所必需的：中间结构域高度保守， 主要负责RNA识别；C-末端结构域可能起作用 RNA和蛋白质的相互作用。片段的结构 含有中间和C-末端区域(159个残基)，来源于 用核磁共振方法对嗜热脂肪芽孢杆菌S4进行鉴定。站点- 定向诱变、选择方法、热力学和动力学 测量将用于探索S4-RNA的以下方面 互动： MRNA或rRNA所涉及的保守表面残基的测定 有约束力的； S4结合与折叠的方式的比较 核糖体和信使RNA识别结构域，特别是 重视镁离子在促进RNA折叠和蛋白质合成中的作用 有约束力的； 通过对S4和S4的研究解析翻译抑制机制 30S亚基与mRNA核糖体结合部位的相互作用； S4与其他核糖体蛋白(S20)协同作用的研究 和S16)与16S rRNA 5‘结构域组装。 这些研究的结果应该有助于对 蛋白质-RNA识别机制和RNA折叠以及阐明 S4在翻译调控和核糖体组装中的特定作用。

Complexes of RNA and protein (RNPs) carry out many essential functions in the cell, including translation. How proteins within these complexes recognized specific sites, promote RNA folding, and contribute to function are important questions. The goal of this project is to explore these question for a ribosomal protein, S4, that has dual functions: it is a key protein in ribosome assembly, binding and organizing a approximately 500 nucleotide domain of the small subunit RNA, and also binds a 110 nucleotide pseudoknot structure in the alpha mRNA, where it represses translation by a novel "entrapment" mechanism. The protein is organized into at least three regions: an N-terminal segment (42 residues) is not required for ribosome assembly: a middle domain is highly conserved and mostly responsible for RNA recognition; a C-terminal domain may contribute to both RNA and protein interactions. The structure of a fragment containing the middle and C-terminal regions (159 residues), derived from Bacillus stearothermophilus S4, will be determined by NMR methods. Site- directed mutagenesis, selection approaches, and thermodynamic and kinetic measurements will be used to explore the following aspects of S4-RNA interactions: Determination of conserved surface residues involved in mRNA or rRNA binding; Comparison of the ways in which S4 binding is linked to folding of the ribosomal and messenger RNA recognition domains, paying particular attention to the role of Mg/2+ in promoting RNA folding and protein binding; Resolution of the translational repression mechanism by studies of S4 and 30S subunit interactions with the mRNA ribosome binding site; Studies of the cooperatively between S4 and other ribosomal proteins (S20 and S16) assembling with the 16S rRNA 5' domain. Results from these studies should contribute to a basic understanding of protein-RNA recognition mechanisms and RNA folding, as well as elucidation of specific S4 roles in translational regulation and ribosome assembly.