Skripkin Ea

Transition of the mRNA sequence downstream from the initiation codon into a single-stranded conformation is strongly promoted by binding of the initiator tRNA

Abstract Using an RNA footprinting technique, accessible sites on the mRNA initiation region bound to the ribosome have been determined. Chemical probing experiments have been done both in the presence and absence of the initiator tRNA with dimethyl sulfate, kethoxal and carbodiimide as reagent probes. As an mRNA, a mini-mRNA containing the initiation region of bacteriophage λ gene cro has been used. This region is characterized by a long single-stranded Shine-Dalgarno (SD) sequence followed by two hairpin structures of which the first one comprises in its loop the initiation codon. As compared to a free mRNA, the only nucleotides additionally protected in the binary mRNA-ribosome complex have been those which belong to the S-D sequence and the initiation codon. The protection of other nucleotides has not changed. Addition of the initiator RNA results in the modification of nucleotides in the stems of the downstream hairpin structures of the initiation region. This reflects their transition into a single-stranded conformation promoted by tRNA. A possible implication of these findings for the decoding process is discussed.

Synthesis and pathway of Luciola mingrelica firefly luciferase in Xenopus laevis frog oocytes and in cell-free systems

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.

The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli

The kinetics ofPhotinus pyralis andLuciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible λPr promoter inEscherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay.E. coli cells were cultivated at temperature schemes 28–42–21°C or 28–21°C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42°C) followed by the incubation at 21°C, for 10 h resulted in the maximal amount of the luciferase protein of 4–5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21°C and reached the maximum after 50–60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.