Regulation of tRNA Gene Expression

不同tRNA的相对丰度受内部控制区(ICR)的序列和间距、5‘和3’侧翼序列以及单个tRNA基因的拷贝数的静态调节。一种蛋白质因子，TFIIIC，核转录复合体的形成。已知ICR中规范序列和间距的变化会影响这种相互作用，这可能决定哪些基因表达。我们参与了光敏交联剂5-N3-脱氧尿苷-5‘-三磷酸(AdUTP)的开发，它可以代替胸苷被酶结合到DNA中。光活性DNA能够与TFIIIC发生序列特异性的相互作用，并可用于鉴定该蛋白与ICR的B-块接触的亚基。我们建议研究该蛋白质与tRNA基因的相互作用，以确定哪些T残基与TRIIIC密切相关，A&Amp；B区块是否与相同或不同的亚基接触，编码序列或侧翼序列中的其他T残基是否可以与该因子交联，以及蛋白质的哪些区域与ICR接触。基于纯化多肽的部分氨基酸序列分析，将使用合成的DNA探针在酵母基因组库中鉴定DNA结合亚基(S)的基因(S)。对该基因(S)的核苷酸序列进行测定，推导出纯化多肽的氨基酸序列分析。将该基因(S)的核苷酸序列进行测定，推导出氨基酸序列。TFFIIIC与AdUTP取代DNA的光交联肽图谱将使我们能够识别蛋白质中与每个tRNA控制区密切接触的位置。影响tRNA基因表达的5‘-侧翼和反密码子序列突变的研究也将完成。这些研究涉及控制转移RNA(TRNA)形成的因素，tRNA是参与将氨基酸转移到新生肽链的核糖核酸分子。其中一个因素是一种名为TFIIIC的蛋白质，它与编码这些tRNA的基因发生反应。研究将使用一种碱基来替代DNA中的胸腺嘧啶，并在光存在的情况下与TFIIIC反应。通过这种方式，可以确定TFIIIC与DNA上的哪个部位发生反应。

The relative abundance of different tRNAs is statically regulated by the sequence and spacing of internal control regions (ICR), both 5' and 3' - flanking sequences, and the copy number of individual tRNA genes. A protein factor, TFIIIC, nucleates formation of transcription complex. Variations from canonical sequences and spacing in the ICR are known to affect this interaction which may determine which genes are expressed. We have participated in development of a photosensitive cross-linking reagent, 5-N3- deoxyuridine-5'-triphosphate (AdUTP), which can be enzymatically incorporated into DNA in place of thymidine. The photoactive DNA is capable of sequence-specific interaction with TFIIIC and can be used to identify the subunit of this protein which contacts the B-block of the ICR. We propose to study the interactions of this protein with tRNA genes to identify which T residues are in close association with TRIIIC, whether A & B blocks contact the same or different subunits, whether other T residues in the coding or flanking sequences can be cross-linked to the factor, and what regions of the protein contact the ICR. The gene(s) for the DNA binding subunit (s) will be identified in a yeast genomic library using synthetic DNA probes based on partial amino acid sequence analysis of purified polypeptides. The nucleotide sequence of this gene (s) will be determined to deduce the amino acid sequence analysis of purified polypeptides. The nucleotide sequence of this gene (s) will be determined to deduce the amino acid sequence. Peptide mapping of TFFIIIC photocrosslinked to AdUTP substituted DNA will allow us to identify sites in the protein which are in close contact with each tRNA control region. Studies of mutations in 5'-flanking and anticodon sequences which affect tRNA gene expression will also be completed. These investigations deal with the factors that control the formation of transfer RNA's, (tRNA's), which are ribonucleic acid molecules involved in transferring amino acids to nascent peptide chains. One of these factors is a protein called TFIIIC that reacts with the genes that code for these tRNA's. The investigations will employ a base that will substitute for thymine in DNA and react with TFIIIC in the presence of light. In this way, the site on the DNA with which TFIIIC reacts can be identified.