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 它的失活不存在唯一的靶mRNA 开始腐烂 相反，有一种核糖核酸内切酶 切割某些嘧啶-腺苷（pyr-A）键。 这些 目标被相关的核糖体保护。 最初的分裂 由于统计原因，通常靠近5 '端， 酶“切掉”大的寡核苷酸片段（可能30至40个）。 200个核苷酸）作为最后一个核糖体 向下传递消息。 碎片被降解为 其他几种RNA酶的单核苷酸酶。 该提案的最初目标是纯化pyr-A酶 使用各种色谱分离。 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 以及其酶学的其他特性。 哺乳动物 胰腺RNA酶在极低浓度下也具有特异性， 对于pyr-A键和两种酶将进行比较， 这种活性可能是由一个酶家族编码的 分布在大自然中。 完整氨基酸序列 酶将被确定。 将构建一种菌株，在该菌株中， RNA酶可以被调节。 脱氧核苷酸探针（15-20 核苷酸）将被合成以编码氨基酸序列 并用于在λ文库中纯化其基因， 总大肠coli DNA。 将使用替代程序，如果 必要 主要建设将包括交换一个 受调控的启动子，例如，trp，并取代含有基因的 DNA通过转化和重组进入宿主。 菌株 与温度敏感的合成或酶的活性将 也要建。 mRNA代谢将在新的 将添加其他RNA酶中的菌株和突变， 确定降解的所有步骤。 gal mRNA的5 '-和3'-末端 将确定酵母的相同的基本机制， 降解发生在低级真核生物中。

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.