In vitro translation.

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.

Second phosphorylation site on alpha subunit of eIF-2 in rabbit reticulocyte lysate.

Eukaryotic protein synthesis initiation factor 2, eIF-2, was purified from either hemin-supplemented (translationally active) or hemin-deficient (translationally inactive) rabbit reticulocyte lysate under conditions chosen and demonstrated to preserve the in situ phosphorylation state. Direct analysis of the phosphate content of the alpha-subunit of eIF-2 was determined by chemical analysis of the isolated alpha-subunit or by a combination of vertical slab gel isoelectric focusing and immunoblotting. These results were compared with those obtained from an indirect analysis utilising the incorporation of [gamma-32P]ATP into eIF-2 by the heme-sensitive eIF-2 alpha-kinase. All three analyses demonstrate that the phosphorylation site specific for the heme-sensitive kinase is unoccupied in translationally active lysate and 25-30% occupied in translationally inhibited lysate. In addition, both direct analyses support the existence of a second phosphorylation site on the alpha-subunit, not regulated by hemin and distinct from that phosphorylated by the heme-sensitive kinase. Different reticulocyte lysate batches vary with respect to the activity of the kinase responsible for phosphorylation of the second site. Further investigations demonstrated that this kinase is a membrane-associated protein.

Indirect inactivation of eukaryotic initiation factor 2 in reticulocyte lysate by selenite.

Addition of selenite to rabbit reticulocyte lysate produces a biphasic pattern of translational inhibition. Sucrose density gradient shows that the onset of translational inhibition is accompanied by decreased Met-tRNAf binding to 43 SN ribosomal subunits and loss of polysomes. Control rates of translation are restored by the addition of exogenous eukaryotic initiation factor 2 (eIF-2). Selenite also directly inhibits Met-tRNAf binding activity of eIF-2. While selenite could react directly with unpaired cysteine residues of eIF-2 to inhibit protein synthesis initiation, a more complex mechanism than a direct inactivation of eIF-2 is suggested by the following observations: 1) translational inhibition produced by selenite is accompanied by an apparent increase in the phosphorylation state of eIF-2alpha; and 2) the extent of translational inhibiton is not proportional to steady-state level of phosphorylation. Rather, the time required for the onset of translational inhibition decreases as the level of eIF-2alpha phosphorylation is increased. This suggests a multistep sequence for eIF-2 inactivation, dependent upon an initial activation of eIF-2alpha kinase and followed by additional eIF-2 modification(s).