The regulation of protein synthesis in mammalian cells VI. Soluble and polyribosome associated components in controlling in vitro polypeptide initiation in HeLa cells

Abstract

The in vitro initiation of polypeptides on endogenous polyribosomes has been studied in extracts from HeLa cells. Regulation of the rate of initiation of polypeptides can be examined. In these experiments an assay using [(35)S]fMet-tRNA(f) (Met) has been developed, and the system further characterized. The system has been separated into a fraction containing polyribosomes with subunits and a fraction containing soluble components. The regulation of initiation has at least two distinct components. There is one factor in the soluble fraction which develops a stimulated response after protein synthesis has been inhibited in intact cells. This stimulation does not require new RNA synthesis during the period of cell "stress."A second component is associated with ribosomes. This factor is necessary for the initiation of polypeptides on endogenous polyribosomes. It disappears gradually when cells are exposed to actinomycin. The disappearance is first manifested by an inability of polyribosomes to respond to stimulated supernatants. This unstable component, which decays in the presence of actinomycin, has no apparent counterpart in systems that measure initiation on exogenous mRNA.