Control of arg-2 Gene Expression in Neurospora

脉孢菌中 arg-2 基因表达的控制

• 批准号: 7585941
• 负责人: MATTHEW Steven SACHS
• 金额: $ 19.66万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585941
• 关键词: Adopted; Affect; Agriculture; Anabolism; Arginine; Bacteria; Complex; Data; Economics; Ejaculation; Enzymes; Exons; Gene Expression; Genetic Translation; Goals; Homologous Gene; In Vitro; Initiator Codon; Lead; Link; Mammals; Mediating; Medical; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Movement; Mutation; Neurospora; Neurospora crassa; Nonsense Codon; Nonsense-Mediated Decay; Open Reading Frames; Peptides; Phenotype; Plant Viruses; Plants; Proteins; Reading Frames; Regulation; Regulator Genes; Ribosomes; Saccharomyces cerevisiae; Scanning; Site; Specific qualifier value; Structure; Terminator Codon; Testing; Translations; Untranslated Regions; Work; Yeasts; animal extract; arginine attenuator peptide; crosslink; follow-up; fungus; in vivo; loss of function; mRNA Decay; mRNA Stability; novel; peptide structure; polypeptide; response

DESCRIPTION (provided by applicant): The goal of this work is to understand the mechanisms by which a nascent peptide encoded by an upstream open reading frame (uORF) controls the movement of ribosomes on mRNA and regulates gene expression. The arginine attenuator peptide (AAP) is encoded by a uORF in the 5'-leader regions of mRNAs specifying a fungal arginine (Arg) biosynthetic enzyme; it reduces gene expression in response to Arg. AAP-mediated regulation is observed in vivo in both Neurospora crassa and Saccharomyces cerevisiae and in vitro, using fungal, plant and animal extracts. The nascent AAP and Arg cause the ribosome to stall. When the AAP functions as a uORF, the ribosome stalls at the termination step. The stalled ribosomes block scanning ribosomes, decreasing gene expression by reducing ribosomal access to the downstream reading frame. The AAP also functions as an internal polypeptide domain to stall ribosomes involved in elongation. Our data lead to a regulatory model in which the AAP adopts a conformation in the ribosome that, with Arg, interferes with decoding at the A-site, or with another step crucial for elongation or termination. A second aspect of regulation by the AAP is that it controls mRNA stability. Both S. cerevisiae CPA1 and N. crassa arg-2 mRNA levels are affected by nonsense-mediated mRNA decay (NMD). AAP-mediated stalling promotes NMD of the CPA1 mRNA. In the absence of AAP-mediated stalling, NMD of the CPA1 mRNA can be promoted by increased ribosome occupancy of the uORF. These data support a regulatory model in which ribosome stalling at the uORF termination codon in response to Arg destabilizes CPA1 mRNA by increasing the extent of nonsense codon recognition by NMD. This link between AAP-mediated stalling and NMD provides unique opportunities for assessing the cis- and trans-acting elements that contribute to NMD. N. crassa, like many fungi of medical, agricultural, and economic importance, but unlike S. cerevisiae, has clear homologs of elF4AIII, Y14, and Magoh, exon junction complex proteins which are involved in engaging NMD in mammals and are implicated in activating the translation of mRNAs with which they associate. Specific aims are as follows: 1. Elucidate the mechanism of ribosome stalling by analyzing the structure of the AAP through (a) cross-linking the nascent AAP to the ribosome to examine their interactions (b) assessing the conformation of the AAP in the ribosome by comparing it to other nascent chains with known conformations; (c) directly examining AAP structure and how Arg affects it by 2D-NMR. 2. Exploit the regulated expression of yeast CPA1 to test key aspects of the faux-UTR model of NMD and follow up on novel observations that AAP- mediated ribosome stalling triggers NMD. 3. Use new and existing N. crassa strains to determine the functions of Neurospora NMD and EJC factors in translation and mRNA metabolism through the analysis of the phenotypes that result from the loss of these functions.

描述（由申请人提供）：这项工作的目的是了解通过上游开放阅读框（UORF）编码的新生肽控制核糖体在mRNA上的运动并调节基因表达的机制。精氨酸衰减肽（AAP）由UORF编码在指定真菌精氨酸（ARG）生物合成酶的mRNA的5'-Leader区域；它响应ARG降低了基因表达。使用真菌，植物和动物提取物在Neurospora crassa和酿酒酵母以及体外观察到AAP介导的调节。新生的AAP和ARG导致核糖体失速。当AAP充当UORF时，核糖体在终止步骤处停滞了。停滞的核糖体阻断扫描核糖体，通过减少对下游阅读框的核糖体访问来降低基因表达。 AAP还充当了与伸长伸长的失速核糖体的内部多肽结构域。我们的数据导致了一个调节模型，其中AAP在核糖体中采用构象，该核糖体与ARG一起干扰了A位点的解码，或者对伸长或终止的另一步骤至关重要。 AAP调节的第二个方面是它控制mRNA稳定性。酿酒酵母CPA1和Crassa Arg-2 mRNA水平都受到胡说八道介导的mRNA衰变（NMD）的影响。 AAP介导的失速促进CPA1 mRNA的NMD。在没有AAP介导的失速的情况下，CPA1 mRNA的NMD可以通过增加UORF的核糖体占用率来促进。这些数据支持一个调节模型，在该模型中，核糖体在UORF终止密码子响应于ARG的情况下，通过增加NMD的胡说八密码子识别的程度，使CPA1 mRNA破坏了CPA1 mRNA。 AAP介导的失速与NMD之间的这种联系为评估有助于NMD的顺式和反式作用元素提供了独特的机会。 N. Crassa像许多医学，农业和经济重要性一样，但与S. cerevisiae不同，Exon结的复杂蛋白具有清晰的ELF4AIIII，Y14和Magoh的同源物，这些复合蛋白参与哺乳动物中的NMD参与，并涉及与与与之相关的MRNA的翻译。具体目的如下：1。通过（a）通过（a）交联的AAP结构来阐明核糖体停滞的机制，将新生AAP交联到核糖体以检查其相互作用（b）评估其通过将其与其他新生链与已知构型进行比较，以评估核糖体中AAP的构象； （c）直接检查AAP结构以及ARG如何通过2D-NMR影响它。 2。利用酵母CPA1的调节表达来测试NMD的人造-UTR模型的关键方面，并跟进了AAP介导的核糖体停滞触发NMD的新型观察结果。 3。使用新的和现有的乳杆菌菌株来确定神经孢子NMD和EJC因子在翻译和mRNA代谢中的功能，这是通过分析由于这些功能丧失而导致的表型的分析。