Yeast intragenic transcriptional control: activation and repression sites within the coding region of the Saccharomyces cerevisiae LPD1 gene.

Abstract

Though widely recognized in higher eukaryotes, the regulation ofSaccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPDI gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPDI::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYCl::acZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate ofLPDI transcription rather than mRNA degradation or translation. Activation from the DASI region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3-to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.Transcriptional control of the expression of most proteincoding (type II) genes requires cis-acting sequences which lie upstream of the TATA box. The mechanisms by which these sequences and the cognate transcription factors influence the rate of transcription are beginning to be realized (reviewed in reference 66). Less understood are the regulatory sequences of a number of bacterial, viral, and mammalian type II genes that are located within transcribed sequences, within either the 5' untranslated leader sequence, introns, or translated sequences.The location of regulatory sequences within transcribed (downstream) regions places them under a variety of constraints not encountered by upstream elements. The intragenic sequence must perform two roles, specifying a motif for a DNA-binding protein while coding for mRNA and the resultant protein. Furthermore, the passage of the bulky transcriptional machinery after every initiation event results in a variety of disturbances to the coding sequence. These include the overwinding and then underwinding of the DNA (36), disruption of nucleosomes (30), and the potential for interference with the DNA-bound regulatory protein. Regulatory proteins that bind to transcribed sequences must therefore have mechanisms that either allow rapid reestablishment of the DNA-protein complex or modify the transcriptional state of the gene which is maintained after the protein is released. To date, RNA polymerase III-transcribed genes have provided a model for downstream regulation of transcription; h...

Carbon catabolite regulation of transcription of nuclear genes coding for mitochondrial proteins in the yeast Kluyveromyces lactis

“…The genes encoding subunits of either of these latter enzymes may therefore turn out to be candidates for regulation at this branch point of respiratory and fermentative metabolism. In line with this expectation, the promoter of the ScLPD1 gene, encoding one of the subunits of PDH in S. cerevisiae, possesses binding sites for the transcription factors ABF1, CPF1, HAP2/34, GCN4 and RAP1 (Bowman et al 1992;Sinclair et al 1994). Characterisation of the corresponding K. lactis genes, encoding subunits of the PDH and PDC complexes, and elucidation of the extent and mechanism of the carbon sourcecontrol of expression will be of great interest.…”

Transcriptional Repression, a Novel Function for 3' Untranslated Regions

“…Typical examples are represented by the negative element identified in the first exon of the rodent c-fos gene [34], positive and negative elements found in the Saccharomyces cerevisiae lipoamide dehydrogenase type-I gene [35] and a negative element which binds the tumor suppressor WT1 in the insulin-like growthfactor-I receptor gene [36]. In contrast, very few examples of putative regulatory sequences located in 3' UTRs have so far been identified.…”

Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA

“…For quantitation of RP026 mRNA, the amount of signal generated from both mutant and wild-type initiation sites (indicated by brackets) were quantitated for lanes 2 to 4. for the ADHl gene amongst different wild-type yeast strains (Bennetzen and Hall, 1982;Pinto et al, 1992Pinto et al, , 1994. The role of Abflp binding-sites in the expression of a variety of genes has been studied (Buchman and Kornberg, 1990;Della Seta et al, 1990a,b;Dequard-Chablat et al, 1991;Halfter et al, 1989;Hamil et al, 1988;Herruer et al, 1989;Kraakman et al, 1991;Mager and Planta, 1990;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993); however, to our knowledge, this is the first report of a shift in the initiation sites of transcription due to a mutation in an Abflp binding-site. In other studies, promoters have often been used to drive expression of the lacZ reporter gene and the effects of Abfl p binding-site mutations have been analysed by monitoring b-galactosidase activity in cellular extracts.…”

“…Abflp alone is not a potent transcriptional activator, normally requiring the cooperation of other weak activators (Buchman and Kornberg, 1990;Della Seta et a/., 1990b;Halfter et al, 1989;Hamil eta/., 1988;Kraakman et a/., 1991;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993). For example, Abflp binding-sites are often associated with a T-rich sequence, which functions in a variety of yeast promoters as a transcriptional activator (Buchman and Kornberg, 1990;GonCalves et al, 1995;Iyer and Struhl, 1995;Lue et al, 1989;Schultes and Szostak, 1991).…”

“…Abfl p is a highly abundant transcriptional activator and is required for constitutive o r regulated transcription of a large number of genes (Buchman and Kornberg, 1990;Della Seta et al, 1990b;Dhawale and Lane, 1993;GonCalves et al, 1995;Halfter et al, 1989;Hamil et al, 1988;Herruer et al, 1989;Kraakman et al, 1991;Mager and Planta, 1990;Sinclair et al, 1994;Trawick et al, 1992;Willett et al, 1993). For these genes Abflp appears to facilitate the access of other regulatory factors to promoters, thereby mediating their regulatory effects (GonCalves et al, 1995;Trawick et al, 1992;Willett et al, 1993).…”