Translation initiation factor-dependent extracts from yeast Saccharomyces cerevisiae.

Translation initiation factor-dependent extracts are prepared from Saccharomyces cerevisiae strains that have no or reduced activity of a translation initiation factor. Elimination of factor activity can be achieved by deletion of the gene encoding the factor if it is not essential for the survival of the strain. If the gene is essential it is placed under the control of the regulatable GAL1 promoter and its expression is shut off in vivo. Alternatively, a temperature-sensitive mutation can be introduced into the gene and the activity of the gene product eliminated in vitro by preincubation of the extract at the nonpermissive temperature. Factor-dependent extracts can be complemented in vitro with purified initiation factor preparations isolated from S. cerevisiae or from Escherichia coli cells expressing them from plasmid-encoded constructs. To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags. Initiation factor-dependent extracts can be used to study initiation factor structure-function relationships and initiation factor requirements for specific mRNA translation.