Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems

A highly efficient mammalian system was developed for the in vitro translation of exogenous rabbit globin messenger RNA. The system consists of purified ribosome subunits from mouse liver, rabbit reticulocytes, or guinea pig brain, partially purified initiation factors from rabbit reticulocytes, and elongation factors, termination factors, aminoacyl-tRNA synthethases and tRNA from rat liver in the form of pH 5-enzymes. (1) Emphasis was put on well-defined, structurally and functionally intact ribosomes, which we found to be the most difficult component of the system in terms of stability of activity. (2) An improved method for extraction of initiation factors from crude reticulocyte ribosomes was developed. Factor preparations of high specific activity were obtained by a simple, partial purification procedure. The crucial point was not to damage the ribosome structure during extraction of the initiation factors and to eliminate inhibitory components during extraction and purification. (3) The efficiency of the system was demonstrated quantitatively by showing that between $\text{3}\text{4}$ and one mRNA molecule per ribosome saturates the system and that each ribosome recycles over the mRNA several times. (4) Major uncertainties and ambiguities in the search for and identification of true initiation factors, as opposed to structural ribosome proteins needed for the reconstitution of damaged ribosomes, can be reduced by using this system.