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.

Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined. To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25. To that end we constructed a hybrid gene consisting of the pertinent 5′‐flanking sequence and the Escherichia coli galK marker gene. The effects on the transcription of this fusion gene of Bal31‐generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells. The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed. Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed. Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element. Moreover, the RPG box functions in both orientations, though not with equal efficiency.

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-7 gene encodes a protein with 71 O/ O amino acid residues identical to the well characterized elongation factor T u of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-7 downstream of a fus homologue and the in vitro activity of Sr.EF-Tul show that tuf-7 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EFTu. Homologues of tuf-7 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tul was present at all times under kirromycin production conditions in submerged and surface-grown cultures of 5. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tul was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of 5. ramocissimus.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.