POLYSOMES AND SUBUNITS--STRUCTURE/FUNCTION RELATIONSHIP

描述(改编自申请人的摘要)：长期目标 这项赠款建议是为了更好地了解催化作用 核糖体RNA在蛋白质合成中的作用。特定的功能已经 在大肠杆菌中被分配到16S和23S rRNAs的许多区域。其目的是 这个项目的目的是更详细地定义这些区域，解密 更高级的结构和功能，通过利用 强大的遗传、生化、生物物理和系统发育技术。这个 最近发现的16S rRNA中影响信使核糖核酸的构象开关 在解码区的通路显示出显著的、动态的性质 核糖体的。这鼓励人们寻找更多的证据 细胞内rRNA不同区域之间的相互作用关系 亚基和亚基之间。主要方法将涉及rRNA。 质粒携带的rrnB操纵子的诱变。不同的寄主菌株和 即使是致命的突变也可以利用质粒载体 表达。第二位点抑制子突变技术(或 加速进化)将被用来识别基本核苷酸 包含功能主题，并发现更远的联系 动态交互的代表。协变位点和碱基 通过系统发育分析鉴定的三联体将被诱变以进一步 定义三维结构，如肽基转移酶区域。 预计这些研究将进一步加深我们对 RRNA在翻译的所有步骤(解码、多肽)中的功能作用 键的形成和移位)，因为它们揭示了 高阶结构和众多的动态过程 定义蛋白质合成。

DESCRIPTION (Adapted from applicant's abstract): The long term objective of this grant proposal is to gain a greater understanding of the catalytic role of ribosomal RNA in protein synthesis. Particular functions have already been assigned to many regions of both 16S and 23S rRNAs in E. coli. The aim of this project is to define these regions in greater detail, deciphering both the higher order structure and function, by utilizing a combination of powerful genetic, biochemical, biophysical and phylogenetic techniques. The recent discovery of a conformational switch in 16S rRNA affecting the mRNA pathway in the decoding region demonstrates the remarkable, dynamic nature of the ribosome. This encourages one to look for additional evidence of interactive relationships between different regions of rRNA within the subunits and between the subunits. The primary approach will involve rRNA mutagenesis of a plasmid-borne rrnB operon. Different host strains and plasmid vectors are available to permit even lethal mutations to be expressed. The technique of second site suppressor mutagenesis (or accelerated evolution) will be utilized to identify essential nucleotides comprising functional motifs and uncover more distant contacts representative of dynamic interactions. Sites of covariation and base triples identified by phylogenetic analysis will be mutagenized to further define three-dimensional structures such as the peptidyl transferase region. It is anticipated that these studies will further our understanding of the functional role of rRNA during all steps of translation (decoding, peptide bond formation and translocation) as they reveal the complex nature of the higher order structure and the multitude of dynamic processes which together define protein synthesis.