METABOLISM OF MESSENGER RNA IN E. COLI

我们将尝试鉴定mRNA的主要5'（和可能的3'）末端， E.乳糖操纵子（lac操纵子）。存在于生长细胞中。 从 早期的研究，我们预期独特的5 '-末端来自起始附近的特定裂解， 在Lac Z和Lac Zy和Ya交界处之间。 特定的lac RNA区域将 纯化并使用γ-32 P-ATP进行5'（或3'）末端标记。 大 在部分RNA酶处理后分离32 P-寡核苷酸， 在2D聚丙烯酰胺凝胶上分离。 他们的位置将由 在它们被洗提并在碱中部分消化之前进行放射自显影， 通过在醋酸纤维素上电泳的2D分离确定的序列 和TLC均相色谱法。 我们正在构建质粒， 这些和其他研究的乳糖操纵子的片段。 同样的方法将 用于鉴定某些lac Oc突变体中lac mRNA的5'末端， 显示了Z信息的衰变和核糖体起始频率的改变。 我们的其他工作表明RNase III参与了乳糖苷酶的加工 留言翻译 将分析来自RNase III细胞的lac mRNA 同样的。

We shall attempt to identify the major 5' (and possible 3') ends of mRNA from the lactose (lac) operon of E. coli that are present in growing cells. From earlier studies we expect unique 5'-ends from specific cleavages near the start of lac z and between the lac zy and ya junctions. Specific lac RNA regions will be purified and 5' (or 3') end labeled using gamma-32P-ATP. Large 32P-oligonucleotides will be isolated after partial RNase treatment and separation on 2D polyacrylamide gels. Their positions will be identified by autoradiography before they are eluted and partially digested in alkali and the sequences determined by 2D separations with electrophoresis on cellulose acetate and TLC homochromatography. We are constructing plasmids that carry specific segments of the lac operon for these and other studies. The same methods will be used to identify the 5' ends of lac mRNA in certain lac Oc mutants which we showed had altered decay and ribosome initiation frequencies for the z message. Other work by us suggested an involvement of RNase III in processing lac z message for translation. The lac mRNA from RNase III-cells will be analyzed similarly.