GENETIC ANALYSIS OF SIGNAL RECOGNITION PARTICLE FUNCTION

在真核生物中，蛋白质被输出到 细胞外空间，插入质膜，或并入 进入溶酶体，通过一系列有序的膜结合 细胞器沿着路线到达它们的最终目的地。 基于体外 使用重组异源系统的实验，Walter和Blobel 我假设，针对第一个，内质网， 内质网（ER），是由核糖核蛋白，指定的信号 识别颗粒（SRP），其识别疏水的序列。 由蛋白质携带的氨基酸，目的是从细胞质通过 这条路。 SRP由六种不同的多肽和一种 7SL RNA分子，所有这些都是体外活性所需的。 我建议研究SRP承认新生儿的机制， 核糖体上的分泌前蛋白质，首先突变改变其 结构，然后确定这是否以及如何干扰功能， vivo. 这些研究选择了酿酒酵母 因为它很容易被基因操纵。 的 策略将是克隆编码酵母7SL RNA的基因， 体外，并测试与改变的副本转化的细胞中的后果。 第一个关键问题是7SL RNA的功能是否是 在酵母中的生存力所必需的，即是致死基因的无效等位基因 到细胞？ 随后，将产生更多的细微病变， 剖析7SL分子的功能。 一旦发生点突变， RNA已经被分离出来，将有可能识别出其他成分， 通过选择第二位点与该分子相互作用的细胞 分泌缺陷的抑制剂。 正确的蛋白质区室化对细胞至关重要; 分泌缺陷在人体内产生病理状态，例如I细胞 疾病和α-1抗胰蛋白酶缺乏。 由于分泌途径 在进化过程中似乎高度保守，这是我的结果 拟议中的实验应该能对最初的步骤提供有价值的见解 这一过程在高等真核生物中，遗传方法是不可能的。 可行

In eukaryotic organisms, proteins which are to be exported to the extracellular space, inserted into the plasma membrane, or incorporated into lysosomes, pass through an ordered series of membrane-bounded organelles along the route to their final destinations. Based on in vitro experiments using a reconstituted heterologous system, Walter and Blobel have postulated that targeting to the first of these, the endoplasmic reticulum (ER), is mediated by a ribonucleoprotein, designated Signal Recognition Particle (SRP), which recognizes a sequence of hydrophobic amino acids carried by proteins destined for export from the cytoplasm via this pathway. SRP is composed of six distinct polypeptides and one molecule of 7SL RNA, all of which are required for activity in vitro. I propose to examine the mechanism by which SRP recognizes nascent pre-secretory proteins on ribosomes by first mutationally altering its structure and then determining whether and how this perturbs function in vivo. The yeast Saccharomyces cerevisiae has been chosen for these studies because of the ease with which it can be manipulated genetically. The strategy will be to clone the gene encoding yeast 7SL RNA, mutagenize it in vitro, and test the consequences in cells transformed with altered copies. The first critical question will be whether the function of 7SL RNA is essential for viability in yeast, i.e. is a null allele of the gene lethal to the cell? Subsequently, more subtle lesions will be generated in order to dissect the function of the 7SL molecule. Once point mutations in this RNA have been isolated, it will be possible to identify other components in the cell which interact with this molecule by selecting for second-site suppressors of the secretory defects. Correct protein compartmentalization is of critical importance to cells; defects in secretion produce pathological states in humans, e.g. I-cell disease and Alpha-1 anti-trypsin deficiency. Since the secretory pathway appears to be highly conserved through evolution, the results of my proposed experiments should provide valuable insight into the initial steps of this process in higher eukaryotes, where a genetic approach is not feasible.