METABOLISM OF MESSENGER RNA IN E. COLI

最近，我们提出了以下mrna降解模型。 原核生物。它的失活没有唯一的靶基因 和腐烂的开始。相反，有一种内切核糖核酸酶 这会裂解某些嘧啶-腺苷(PYR-A)键。这些 靶标受到相关核糖体的保护。最初的卵裂 出于统计原因，通常在5‘端附近 酶“砍掉”大的寡核苷酸片段(可能是30到 200个核苷酸)从新产生的5‘端作为最后一个核糖体 继续向下传递消息。碎片被降解为 由其他几种核糖核酸酶产生的单核苷酸。 这项提议的最初目标是提纯PYR-A酶 使用不同的层析分离。到目前为止，我们已经 以5S rRNA为底物，观察其比活性。 将合成底物以进一步确定其特异性 以及它的其他酶学特性。哺乳动物 极低浓度的胰腺核糖核酸酶也具有特异性 对于PYR-A键和两种酶将进行比较的观点 这种活动可能是由一系列酶编码的 分布在整个自然界。完整的氨基酸序列 酶的含量将会被测定。 将构建一种菌株，在该菌株中合成或活性 核糖核酸酶是可以调节的。脱氧核苷酸探针(15-20 核苷酸)将被合成以编码氨基酸序列 并用于纯化该酶的Lambda文库中的基因 总的E.Coli DNA。如果出现以下情况，将使用替代程序 这是必要的。主要的建设将包括交换一个 调控启动子，例如色氨酸，并取代含有基因的 DNA通过转化和重组进入宿主。菌株 具有温度敏感型合成或活性的酶将 也将被建造。信使核糖核酸的代谢将在新的 将添加其他核糖核酸酶的菌株和突变，以便 确定降级过程中的所有步骤。Gal基因的5‘端和3’端 将被鉴定为酵母菌，看看同样的基本机制 退化发生在较低的真核生物中。

Recently, we proposed the following model for mRNA degradation in procaryotes. There is no unique target mRNA for its inactivation and initiation of decay. Instead, there is an endoribonuclease that cleaves certain pyrimidine-adenosine (pyr-A) bonds. These targets are protected by associated ribosomes. An initial cleavage is usually near the 5'-end for statistical reasons and the same enzyme "chops off" large oligonucleotide fragments (perhaps 30 to 200 nucleotides) from the newly-created 5'-end as the last ribosome progresses down the message. The fragments are degraded to mononucleotides by several other RNases. The initial goal of this proposal is to purify the pyr-A enzyme using various chromatographic separations. So far, we have observed its specific activity using 5S rRNA as a substrate. Substrates will be synthesized to define further its specificity as well as other characteristics of its enzymology. Mammalian pancreatic RNase at very low concentrations also has a specificity for pyr-A bonds and the two enzymes will be compared with a view that this activity may be encoded by a family of enzymes distributed throughout Nature. The complete amino acid sequence of the enzyme will be determined. A strain will be constructed in which the synthesis or activity of the RNase can be regulated. Deoxynucleotide probes (15-20 nucleotides) will be synthesized to code for amino acid sequences of the enzyme and used to purify its gene in a lambda library of total E. coli DNA. Alternative procedures will be used, if necessary. The major construction will include exchanging a regulated promoter, e.g., trp, and substituting the gene-containing DNA into the host by transformation and recombination. Strains with temperature-sensitive synthesis or activity of the enzyme will also be constructed. mRNA metabolism will be studied in the new strains and mutations in other RNAse would be added in order to identify all steps in degradation. The 5'- and 3'-ends of gal mRNA of yeast will be identified to see if the same basic mechanism of degradation occurs in a lower eucaryote.