Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12‐nucleotide‐long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5′‐flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF‐UASrpg binding by analysing–using footprinting and gel electrophoretic retardation techniques–the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half‐life of the protein‐DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus.

Shifting a yeast culture from an ethanol-based medium to a glucose-based medium causes a coordinate increase of the cellular levels of ribosomal protein mRNAs by about a factor 4 within 30 min. Making use of hybrid genes encompassing different portions of the 5'-flanking region of the L25-gene, we could show that the increase in mRNAs is a transcriptional event, mediated through DNA sequences upstream of the ribosomal protein (rp) genes. Further analysis revealed that sequence elements are involved that many rp-genes have in common and that previously were identified as transcription activation sites (RPG-boxes or UASrpg). Using appropriate deletion mutants of the fusion genes we could demonstrate that a single RPG-box is sufficient for the transcriptional upshift. In addition, both copy genes encoding rp28 which differ considerably in their extent of transcriptional activity, show the upshift effect in a proportional manner. Definite proof for the role of the UASrpg in nutritional regulation was obtained by examining the effect of a synthetic RPG-box on transcription.

Transcription of the gene encoding yeast ribosomal protein L25 was previously shown to be activated through tandemly arranged upstream sequence elements that most rp-genes in yeast have in common. A single copy of such a conserved element is now demonstrated to restore transcription of an inactivated heterologous gene, which confirms its role as a genuine UAS: UASrpg. Though a single box is sufficient to activate transcription, most rp-genes harbor two neighbouring elements. Northern analysis of mutants of the L25 upstream region lacking either the gene-distal (RPG1) or the gene-proximal (RPG2) box provided evidence that RPG2 is significantly more effective than RPG1 in vivo. Moreover the sum of the effects of the individual boxes as measured separately is significantly lower than their joint effect, supporting cooperative interaction between the two boxes in vivo. Making use of oligomer-insertion experiments several additional features of the UASrpg were elucidated. First of all we confirmed that the extent of transcription activation by the UASrpg depends upon the orientation of the element. Secondly we show that a certain minimal distance (greater than 100 n) between UASrpg and the transcription initiation site is required for transcription activation. Finally, internal deletion of the L25-upstream region as well as oligomer-insertion shed some light on the nucleotide requirements of the UASrpg.

Using a heterologous probe containing a fragment of the L25-gene from Saccharomyces carlsbergensis we have isolated a DNA-fragment of Candida utilis carrying the gene encoding ribosomal protein L25. This gene is present in a single copy on the C. utilis genome, though as two distinguishable alleles. Both alleles have been isolated and sequenced including their flanking regions. The nucleotide sequence of the amino acid coding region of the C. utilis gene turned out to be highly homologous (83%) to the L25-gene of S. carlsbergensis. At the protein level the degree of homology is about 87%. Codon usage in both organisms appears to be somewhat different. Just like the Saccharomyces gene, the L25 gene in C. utilis appears to be split in its 5th codon, though the identity of this codon has changed. Intron as well as 5'- and 3'-flanking sequences have almost completely diverged, with some notable exceptions. Of the intervening sequences the 5'- and 3'-splice sites as well as the putative lariat branch site are conserved. In the 5'-flanking region, at a distance of about 330 n from the initiation codon, a conserved nucleotide element is present that is very similar to the upstream transcription activation site previously found in front of the ribosomal protein genes in Saccharomyces.