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A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.

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Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or I-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region.Transcription of several well-studied genes in Saccharomyces cerevisiae is thought to require at least two cisacting regions of DNA: the proximal TATA element (12,23,32,34) and the distal upstream activating sequence (UAS;9,14,23,25,45,46,50). UASs are bidirectional elements: they function even when inverted (22,36,47). Transcription does not always require the presence of a TATA element; efficient expression of the PGK gene depends on UASPGK but does not require TATA sequences (36). Unlike mRNA synthesis in higher eucaryotes, sequences in the vicinity of each mRNA start site contribute to the specificity of transcription initiation (12,32,34). Interestingly, some DNA sequences may activate transcription by virtue of their unique structural properties: poly(dA-dT) homopolymer tracts can function as bidirectional promoter elements in the yeast genome (48). These regions have a helix repeat of 10.0 base pairs (bp) instead of the normal 10.6 bp (38,40) and are associated with bends in DNA (28, 31).Of primary concern when attempting to analyze the yeast genome comprehensively by using promoter libraries is the large number of yeast colonies (approximately 106 [43]) that must be screened. The use of two reporter genes (divergently flanking the cloning site) should reduce that number by 50% relative to libraries that use a single reporter gene. A vector that contains two reporter genes permits the detection of a unidirectional promoter regardless of its orientation in the cloning site. Also, if a promoter is bidirectional, it can be isolated from the library in a configuration that allows the simultaneous monitoring of transcription in both directions along the DNA helix. With this in mind, we have used a promoter-cloning vector (pPCO; Fig. 1 uninterrupted coding region. The 53-bp upstream intergenic region lacks a UAS, poly(dA-dT) tract, or TATA element.We created a comprehensive S. cerevisiae genomic library in pPCO (43) by inserting random fragments into the adhllac4 intergenic region. Filter colony assays were used to detect eithe...

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