Khaitovich P, Mankin A S, Green R, Lancaster L, Noller H F
Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):85-90. doi: 10.1073/pnas.96.1.85.
Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from T. aquaticus rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA "cage" surrounding a core of a subset of ribosomal proteins.
嗜热水生栖热菌核糖体的肽基转移酶活性对通过蛋白酶消化、十二烷基硫酸钠（SDS）和苯酚抽提去除大量核糖体蛋白具有抗性。为了确定肽基转移酶所需的大分子成分数量上限，对通过抽提嗜热水生栖热菌大核糖体亚基获得的颗粒进行了分离，并对其RNA和蛋白质组成进行了表征。有活性的亚核糖体颗粒含有23S和5S rRNA，以及与大量8种核糖体蛋白相关联的成分。对这些蛋白质进行N端测序后确定它们为L2、L3、L13、L15、L17、L18、L21和L22。未发现之前认为对形成有活性的肽键颗粒的重组必不可少的核糖体蛋白L4。这些发现与之前的重组实验结果一起，将肽基转移酶中心可能的必需大分子成分数量减少到23S rRNA以及核糖体蛋白L2和L3。从嗜热水生栖热菌rRNA中完全去除核糖体蛋白会导致颗粒三级折叠的丧失以及肽基转移酶的失活。经核糖核酸酶处理后，有活性的亚核糖体颗粒中的蛋白质对蛋白酶水解的可及性显著增加。这些结果以及50S核糖体亚基比30S亚基对SDS抽提表现出更高抗性的观察结果，与所提出的50S亚基结构组织相符，该组织涉及围绕核糖体蛋白子集核心的一个RNA“笼”。
