ACTIVITY-DEPENDENT POLYPEPTIDE ELONGATION IN MUSCLE

该项目的直接目标是确定新生儿的减少是否 多肽延伸率导致蛋白质合成的快速下降 在非承重骨骼肌中观察到。明确确定 肌肉萎缩初始事件的作用部位将有助于 定义维持正常基因表达的细胞内信号。 这些信号可能会影响骨骼肌对慢性应激的适应 卧床休息、截瘫、肢体固定和失重。 大鼠比目鱼肌失去80%的肌原纤维蛋白，如果阻止 负重 尽管所有水平的基因表达都受到影响， 最快速的变化发生在翻译层面;在五个小时内， 非承重肌原纤维蛋白合成速率的开始 至少减少25%，最终下降50%。 初步数据表明，最初的缺陷可能是一个减少率 新生的多肽合成，表现为多聚核糖体的增加， 尺寸 这项建议的工作假设是， 延伸引起80 S核糖体在mRNA上的积累。 的 实验还将确定这种形式的调节是否针对 收缩蛋白 两个实验将测试更多的80 S核糖体， mRNA。 第一个将测量放射性氨基酸的含量 进入新生的多肽库。 校正氨酰-tRNA特异性 活性，该值与新生多肽的数量成比例 (and因此80 S核糖体的数量）。 的 第二个实验将直接测量核糖体亚基的数量 与α-肌动蛋白和细胞色素c mRNA相关。 为了测试 调节是特异性的收缩蛋白，非肌肉基因（β- 半乳糖苷酶和醇脱氢酶）将组成性表达 通过逆转录病毒介导的基因转移来治疗萎缩的肌肉。 的mrna 因为这些基因不会反映多核糖体相对于α的大小变化， 肌动蛋白，如果调节是特异性的收缩蛋白。

The immediate goal of this project is to determine if a decrease in nascent polypeptide elongation rate causes the rapid decrease in protein synthesis observed in non-weightbearing skeletal muscle. A clear identification of the site-of-action of the initial events in muscle atrophy will help to define the intracellular signals that maintain normal gene expression. These signals may influence the adaptation of skeletal muscle to chronic bedrest, paraplegia, limb immobilization, and weightlessness. Rat soleus muscle loses 80% of its myofibril protein if prevented from weightbearing. Although all levels of gene expression are affected, the most rapid changes occur at the level of translation; within five hours of the onset of non-weightbearing myofibril protein synthesis rate has diminished by at least 25% as it evolves toward an eventual 50% decline. Preliminary data indicate that the initial defect may be a decreased rate of nascent polypeptide synthesis that is manifest as in increased polysome size. The working hypothesis of this proposal is that a slowed rate of elongation causes an accumulation of 80S ribosomes on the mRNA. The experiments will also determine if this form of regulation is specific for contractile proteins. Two experiments will test for more 80S ribosomes per mRNA. The first will measure amount of radioactive amino acid incorporated into the nascent polypeptide pool. Corrected for aminoacyl-tRNA specific activity, this value is proportional to the number of nascent polypeptides (and hence the number of 80S ribosomes) associated with the mRNA. The second experiment will directly measure the number of ribosomal subunits associated with both alpha-actin and cytochrome c mRNAs. To test if the regulation is specific for contractile proteins, non-muscle genes (beta- galactosidase and alcohol dehydrogenase) will be constitutively expressed in the atrophying muscle by retroviral-mediated gene transfer. The mRNAs for these genes will not reflect the polysome size shift relative to alpha- actin if regulation is specific for contractile proteins.