Mutational analysis of the upstream activation site of yeast ribosomal protein genes

Most ribosomal protein (rp-)genes in yeast are preceded by conserved sequence motifs that act as upstream transcription-activating sites (RPG box). These sequence elements have previously been shown to represent specific binding sites for a protein factor, TUF. Comparison of the various nucleotide elements identified so far indicates a remarkably high degree of variation in the respective sequences. On the other hand, a methylation interference study performed with one RPG box revealed close contact points with the TUF protein along the entire sequence. To investigate the sequence requirements of the RPG box, we inserted synthetic oligonucleotides that differed from the general consensus sequence ACACCCATACATTT at single positions into a deletion mutant of the L25 promoter that lacked its natural RPG elements. Transcription activity was estimated by Northern analyses of the cellular level of L25-galK hybrid transcripts. The results show that in the 3' part of this sequence element single substitutions are allowed at all positions, in the 5' part, however, the nucleotide requirements appear to be more stringent. In particular, the invariant C at position 5 of the consensus sequence is absolutely necessary for its enhancer function.