Initiation of hemoglobin synthesis: comparison of model reactions that use artificial templates with those using natural messenger RNA.
Centrifugation, Density Gradient
Peptide Chain Elongation, Translational
Peptide Chain Initiation, Translational
PARTIAL REACTIONS DESIGNED TO STUDY THE INDIVIDUAL STEPS IN THE INITIATION OF PROTEIN SYNTHESIS MAY BE DIVIDED INTO TWO GROUPS: artificial models using artificial mRNA templates and natural models using naturally occurring mRNA. The requirements for each of the reticulocyte initiation factors, IF-M(1), IF-M(2), and IF-M(3), and for GTP are examined using these models in order to determine if either type of model is a valid representation of the events occurring in natural initiation. Binding of the initiator tRNA, Met-tRNA(F), to endogenous mRNA requires IF-M(1), IF-M(2), and GTP. A requirement for hydrolysis of GTP is not found when the artificial template ApUpG is used since GDPCP may be substituted for GTP. Met-tRNA(F) bound to the template ApUpG by IF-M(1) + IF-M(2) can form a peptide bond with the aminoacyl-tRNA analog, puromycin. Met-tRNA(F) bound by IF-M(1) + IF-M(2) to the initiator codon of natural globin mRNA, however, cannot form a peptide bond with either puromycin or valine-tRNA unless IF-M(3) is also present. The requirements for Met(F)-valine synthesis on exogenous globin mRNA are the same as the requirements on endogenous mRNA. Synthesis of the initial tripeptides of the alpha and beta chains of rabbit globin, Met(F)-Val-Leu and Met(F)-Val-His, requires, in addition, leucyl-tRNA and histidyl-tRNA. It appears, therefore, that model systems that use natural messenger RNA can duplicate the factor and energy requirements of natural initiation, but that the model systems thus far studied that use artificial messengers as templates do not.