Kick Maurer

C‐terminal domains of general regulatory factors Abf1p and Rap1p in Saccharomyces cerevisiae display functional similarity

Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C‐terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p–Rap1p and the Rap1p–Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T‐rich promoter element. These data strongly suggest that the C‐terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C‐terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.

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

SummaryMost 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.

Structure of the ABF1-homologue from Kluyveromyces marxianus.

By transformation of a Saccharomyces cerevisiae mutant strain conditionally expressing the ABF1-gene, a Kluyveromyces marxianus DNA fragment carrying the gene encoding the ABF1-homologue of this yeast strain (KmABF1) was selected. Comparison of the sequence of the KmABF1 gene with that encoding Saccharomyces cerevisiae ABF1 and the previously isolated ABF1-gene from Kluyveromyces lactis (KlABF1) revealed distinct regions displaying considerable homology and therefore most likely representing sequences encoding essential domains. In addition to the domains putatively involved in DNA binding of the protein factor, two short conserved amino acid sequence elements at the C-termini of the homologous proteins were identified, which are proposed to play a part in their trans-acting functions. This is the first report on the structure of a regulatory protein factor from K. marxianus.

18 Identifying Stress Genes

Publisher Summary This chapter focuses on identifying stress genes. Any unfavorable circumstance that adversely affects growth can be designated as a stress. Such circumstances differ widely and include not only environmental challenges such as heat or cold shock, osmotic dehydration, salt stress, ethanol stress, the extremes of pH, heavy metal challenges, or oxidative stress, but also the conditions of nutrient limitation. The chapter discusses the trans -acting factors that are known to mediate stress-responsive transcriptional activation and elaborates the stress signaling pathways elucidated so far that influence the activity of these transcription factors. The term “stress-responsive” is applied only to those genes whose transcription is elevated after stress exposure. The chapter presents the resultsthat were obtained with a grid filter of RNAs from cells exposed to different stresses. This promises to be a valuable experimental tool, both for examining the pattern of expression of well-known stress-responsive genes and for rapidly revealing the stress-induction patterns of novel stress-responsive genes. Apart from stress-responsive genes (genes whose transcription is increased upon stress), many other genes also play a part in the stress response without themselves being stress-inducible. The chapter also briefly discusses possible experimental strategies whereby these latter stress genes might be detected.

Methanol metabolism in Paracoccus denitrificans I. Isolation and characterization of mutants with defects in methanol utilization

Bacillus licheniformis is a facultative aerobic bacterium used in the fermentation industry for the production of enzymes. For a complete description of the production process knowledge of growth parameters, electron-chain composition and efficiency of energy conservation is obligatory. B. licheniformis S1684 is able to produce an alkaline serine protease exocellularly. In glucoselimited chemostat cultures the specific rate of protease production was maximal at a specific growth rate value (Ix) of 0.22. At higher growth rates protease production was repressed (catabolite repression). Dependent on Ix 10-20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products (Tp) was 4.1, showing that products are not more reduced than biomass. Maximum molar growth yields were high and indicated a high efficiency of growth. The values of yg~auX and y ~ x were 83.8 and 53.3, respectively. When y~ax was corrected for the amount of glucose used for product formation a value of 100.3 could be calculated. These high maximum YATP. molar growth yields most probably are caused by a high max In anaerobic batch experiments a YATP of 14.6 could be determined. The efficiency of energy conservation most probably is low, which is indicated by the measured H +/O ratios of about 4 in cells taken at different Ix-values during glucose limitation, indicating that at the most one or two traditional sites of oxidative phosphorylation are active. The strain used appeared to be instable in protease production and cell morphology. Non-protease-producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells are relaxed (tel-) and non-producing cells stringent (rel § In non-protease-producing chemostat cultures y~ax was significantly higher because no glucose was needed for protease production.