Essential Features of the Peptidyl Transferase Center in theYeast Mitochondrial Ribosome

在大内糖体亚基的肽基转移酶中心（PTC）形成肽键是所有核糖体的主要催化活性。了解PTC的结构和催化机制是核糖体研究领域的一个重要目标。PTC中的大亚基核糖体RNA （large subunit ribosomal RNA, LSU rRNA）的序列和结构在自然界中是高度保守的，有大量证据支持rRNA可能在催化PT反应中起到核酶的作用。目前的观点认为r蛋白在维持rRNA中活性位点的构象方面具有结构作用，尽管r蛋白的催化作用尚未完全排除。利用酵母线粒体核糖体作为实验系统，目前的项目重点研究了四种细菌PTC蛋白（L2, L3， L16和L27）和rRNA核糖甲基转移酶（Pet56p）的线粒体同源物的分子遗传学分析，该酶催化LSU rRNA PTC中普遍保守的G核苷酸上2' O甲基鸟苷的形成。大肠杆菌核糖体的模型表明，修饰的核苷酸在rRNA中的重要性，其中大约24个修饰的核苷酸大部分聚集在核糖体功能中心的mE~NA tRNA肽复合物周围。大肠杆菌23S rRNA中的三个修饰是在结构域v的PTC高度保守的核苷酸上的核糖甲基化。在其他最小修饰的线粒体LSU rRNA中保留了三个修饰的核苷酸，这表明这些特殊修饰在核糖体组装或功能中或两者中都起着基本作用。事实上，pet56突变体不能组装54S核糖体亚基。一个很大的分子遗传学方法将用于研究Gm2251在酵母线粒体核糖体中的功能需求。基于酵母PET56对大肠杆菌突变体的功能互补策略将用于鉴定Pet56p的细菌同源基因。这些研究将为核糖体主要催化中心的结构、功能和组装提供新的信息。通过肽键连接氨基酸是生物蛋白质合成的基本反应。该反应发生在大核糖体亚基的肽基转移酶中心（PTC），并被认为在所有细胞中通过相同的催化机制发生。一个重要的目标是了解核糖体RNA （rRNA）和核糖体蛋白在PTC的结构和催化活性中的各自作用。目前的想法倾向于核糖体蛋白在维持rRNA的催化活性位点方面的结构作用，但对潜在的分子结构只有初步的了解。在PTC中有核苷酸修饰的功能要求。最近的研究结果表明，在酵母线粒体核糖体中，在特定G核苷酸的糖上添加甲基（CH3）是形成PTC的必要步骤。这个G核苷酸在所有已知的LSU rna中都是保守的。经过充分研究的大肠杆菌核糖体含有大约24个修饰的核苷酸，它们都聚集在核糖体的功能中心周围。相比之下，酵母线粒体核糖体只有三个修饰的核苷酸，每个核苷酸都与大肠杆菌LSU rRNA的PTC中的一个修饰相同。在功能性LSU rRNA中仅存在三个修饰的核苷酸，强调了这些特殊修饰的潜在重要性。这项研究的主要目的是了解为什么在一个包含3000个核苷酸长的rRNA和大约40个ibosomal protein的巨大的大分子复合物中添加一个甲基是如此重要。总之，这些研究将提供关于核糖体主要催化中心的新信息。* * *

Mason 9419340 The forrnation of peptide bonds in the peptidyl transferase center (PTC) of the large nbosomal subunit is the primary catalytic activity of all ribosomes. An important objective in the field of ribosome research is to understand the structure and catalytic mechanism of the PTC. The sequence and structure of the large subunit ribosomal RNA (LSU rRNA) in the PTC are very highly conserved in nature, and there is abundant evidence supporting the possibility that rRNA acts as a ribozyme in catalyzing the PT reaction. Current thinking favors a structural role for the r proteins in maintaining the conformation of active sites in the rRNA, although a catalytic role for r proteins has not been ruled out completely. Using the yeast mitochondrial ribosome as an experimental system, the current project has focused on the molecular genetic analysis of mitochondrial homologues of four bacterial PTC proteins (L2, L3, L16 and L27) and an rRNA ribose methyltransferase (Pet56p) that catalyzes the formation of 2' O methylguanosine at a universally conserved G nucleotide in the PTC of LSU rRNAs. The importance of modified nucleotides in rRNA is suggested by models of the E. coli ribosome in which the majority of the approximately 24 modified nucleotides are clustered around the mE~NA tRNA peptide complex in the functional center of the ribosome. Three of the modifications in the 23S rRNA of E. coli are ribose methylations at highly conserved nucleotides in the PTC of domain V. The retention of three modified nucleotides in the otherwise minimally modified mitochondrial LSU rRNA points to a fundamental role for these particular modifications in ribosome assembly or function or both. Indeed, pet56 mutants fail to assemble 54S ribosomal subunits. A largely molecular genetic approach will be used to study the functional requirement for Gm2251 in yeast mitochondrial ribosomes. A strategy based on functional complementation of E. coli mutants by yeast PET56 will be used to i dentify the gene for the bacterial homologue of Pet56p. These studies will provide new information about the structure, function and assembly ofthe principal catalytic center ofthe ribosome. %%% Linking amino acids through peptide bonds is the fundamental reaction of biological protein synthesis. This reaction takes place in the peptidyl transferase center (PTC) of the large ribosomal subunit and is thought to occur by the same catalytic mechanism in all cells. An important objective is to understand the respective roles ofthe ribosomal RNA (rRNA) and ribosomal proteins in the structure and catalytic activity of the PTC. Current thinking favors a structural role for the ribosomal proteins in maintaining the catalytic active sites in the rRNA, but there is only a rudimentary understanding of the underlying molecular architecture. There is a functional requirement for nucleotide modification in the PTC. Recent results have shown that the addition of a methyl (CH3) group to the sugar of a specific G nucleotide is an essential step in the formation of the PTC in yeast mitochondrial ribosomes. This G nucleotide is conserved in all ofthe known LSU rRNAs. The well studied E. coli ribosome contains ca. 24 modified nucleotides, all clustered around the the functional center of the ribosome. In contrast, the yeast mitochondrial ribosome has only three modified nucleotides, each identical to one of the modifications in the PTC of E. coli LSU rRNA. The presence of only three modified nucleotides in a functional LSU rRNA underscores the potential importance of these particular modifications. The major objective of this research is to understand why the addition of a single methyl group is so crucial in a huge macromolecular complex containing a 3,000 nucleotide long rRNA and approximately 40 ibosomal proteins. Overall, these studies will provide new information about the principal catalytic center ofthe ribosome. ***