TRANSLATION INITIATION FACTOR-DEPENDENT EXTRACTS FROM YEAST SACCHAROMYCES-CEREVISIAE

Translation initiation factor-dependent extracts are prepared from Sac charomyces cerevisiae strains that have no or reduced activity of a tr anslation initiation factor. Elimination of factor activity can be ach ieved by deletion of the gene encoding the factor if it is not essenti al for the survival of the strain. If the gene is essential it is plac ed under the control of the regulatable GAL1 promoter and its expressi on is shut off in viva. Alternatively, a temperature-sensitive mutatio n can be introduced into the gene and the activity of the gene product eliminated in vitro by preincubation of the extract at the nonpermiss ive temperature. Factor-dependent extracts can be complemented in vitr o with purified initiation factor preparations isolated from S. cerevi siae or from Escherichia coli cells expressing them from plasmid-encod ed constructs. To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags. Initiation f actor-dependent extracts can be used to study initiation factor struct ure-function relationships and initiation factor requirements for spec ific mRNA translation. (C) 1997 Academic Press.