METABOLISM OF MESSENGER RNA IN E. COLI

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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.