U1trastructural Analysis of RNA Synthesis and Processing

9513589 Beyer这项研究涉及真核基因表达中涉及的基本细胞机制的细节，特别是在前mRNA剪接和3'末端形成的水平。 一个不寻常的体内超微结构的方法，“米勒染色质扩散程序”，将被用来分析特定基因的共转录事件后，显微注射到非洲爪蟾卵母细胞核。 人们可以可视化染色体外质粒载体上的特定基因的多个拷贝，并获得关于RNA转录和加工的结构方面的全局信息，以单个基因和单个转录本的分辨率。 研究将集中在组成性剪接内含子上剪接位点识别和剪接体组装的早期阶段。 对于剪接位点如何在典型的长内含子中配对，人们知之甚少。 超微结构的方法已经揭示了剪接位点在其合成的几秒钟内被识别和配对，即使是10-20个内含子的长内含子。 超微结构与这样的可能性一致，即一旦合成，5'剪接位点以某种方式被拴在RNA从聚合酶出现的位点附近，以便与下一个识别的3'剪接位点配对。 实验的目的是测试参与各种顺式和transacting因素的早期内含子识别现象，无论是否拴系参与，也测试拴系机制的可能性。 第二个具体目标是使用这种超微结构测定来确定基因末端的转录物释放机制，并区分多聚腺苷酸化信号和终止/暂停信号对该过程的相对贡献。 在转录活性状态下对基因进行可视化，以确定基因3'端转录物的基因长度和密度，从而产生关于转录物释放位点、转录物释放模式（切割或终止）、转录物释放效率和转录暂停在转录物释放中的可能作用的直接视觉信息。 已经提出了两种可供选择的转录本释放模型，这两种模型都适应了转录本释放对功能性poly（A）信号的已知依赖性（即，在多聚腺苷酸位点的共转录RNA切割相对于聚合酶改变为有终止能力的形式）。这项研究的结果将允许这些模型之间的明确区分。 蛋白质合成的信息编码在散布在没有明确定义功能的RNA序列中的RNA序列块中。 一组蛋白质负责从RNA的其余部分切除氨基酸指定序列的块，并将它们连接成线性链以递送到蛋白质合成复合物。这些研究涉及一种超微结构方法，以可视化该过程的各个步骤，并确定中间体作为确认假定的RNA加工机制的一种手段。 第二个目标是使用相同的测定来确定RNA转录物如何从DNA基因序列的末端释放，以及不同遗传信号在该过程中的作用。 将在活跃转录状态下对基因进行超微结构观察，以获得有关转录机制的机制信息。 * * *

9513589 Beyer This research involves the details of the basic cellular machinery involved in eukaryotic gene expression, specifically at the level of pre-mRNA splicing and 3' end formation. An unusual in vivo ultrastructural approach, the "Miller chromatin spreading procedure", will be used to analyze co-transcriptional events on specific genes after their microinjection into Xenopus laevis oocyte nuclei. One can visualize multiple copies of specific genes on extrachromosomal plasmid vectors and gain global information concerning structural aspects of RNA transcription and processing at the resolution of the individual gene and the individual transcript. Studies will concentrate on the very early stages of splice site recognition and spliceosome assembly on constitutively spliced introns. There is little understanding of how splice sites are paired across typical long introns. The ultrastructural approach has revealed that splice sites are recognized and paired within seconds of their synthesis, even for long introns of 10-20 kilobases. The ultrastructure is consistent with the possibility that, once synthesized, 5' splice sites are somehow tethered near the site of RNA emergence from the polymerase in order to be paired with the next recognized 3' splice site. Experiments are designed to test the involvement of various cis and transacting factors in the early intron recognition phenomenon regardless of whether or not tethering is involved, and also to test the possibility of a tethering mechanism. The second specific objective is to use this ultrastructural assay to determine the mechanism of transcript release at the end of a gene, and to distinguish the relative contributions of polyadenylation signals and termination/pause signals to this process. Genes will be visualized in a transcriptionally active state for determination of the gene length and density of the transcripts at the 3' end of the gene, yielding direct visual information on the site(s) of transcript release, the mode of transcript release (cleavage or termination), the efficiency of transcript release, and the possible role of transcriptional pausing in transcript release. Two alternative models for transcript release have been proposed, both of which accommodate the known dependence of transcript release on a functional poly(A) signal, (i.e., co-transcriptional RNA cleavage at the poly(A) site vs alteration of the polymerase to a termination-competent form). The results of this study will allow a clear distinction between these models. %%% The information for the synthesis of a protein is encoded in blocks of sequences of RNA interspersed within RNA sequences that have no clearly defined function. A group of proteins are responsible for the excising the blocks of amino acid specifying sequences from the rest of the RNA, and joining them in a linear chain for delivery to the protein synthesizing complex. These studies involve an ultrastructural approach to visualize the separate steps of the process, and to identify intermediates as a means of confirming postulated mechanisms of processing the RNA. A second objective is to use the same assay to determine how the RNA transcript is released from the end of a DNA gene sequence, and the role of different genetic signals on this process. Genes will be ultrastructurally observed in an actively transcribed state to allow mechanistic information to be obtained on transcription mechanisms. * * *