Georg Stöffler

Crel, the carbon catabolite repressor protein from Trichoderma reesei

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR‐primers were designed to amplify a 0.7‐bp fragment of the crel gene, which was used to clone the entire gene. It encodes a 402‐amino acid protein with a calculated M r, of 43.6 kDa. Its aa‐sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa‐region containing the C2H2 zinc finger and several aa‐regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa‐region rich in the SPXX‐motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the crel translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (±2) kDa in cell‐free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa‐sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S‐transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5′‐GCGGAG‐3′, which matches well with the A. nidulans consensus sequence for CreA binding (5′‐SYGGRG‐3′). Cell‐free extracts of T. reesei formed different complexes with DNA‐fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins

SummaryRibosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.Ribosomes with an altered L4 protein bound erythromycin rather poorly and the formation of N-acetylphenylalanyl puromycin was drastically decreased. On the other handribosomes with an altered L22 protein bound erythromycin as efficiently as wild type ribosomes and their puromycin reaction was at least as good as that of wild type ribosomes.Transduction experiments showed that the mutations affecting both proteins, L4 and L22, are located very close to the str and spc genes, nearer to the spc than to str gene.

Determination of the location of proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S ribosomal subunit of Escherichia coli by immune electron microscopy

SummaryThe location of the ribosomal proteins L14, L17, L18, L19, L22 and L23 on the surface of the 50S subunit of E. coli ribosomes was determined by immune electron microscopy. Antibodies (bivalent IgGs) specific for the six ribosomal proteins were used to form 50S subunit dimers (50S-IgG-50S). The dimers were separated from non-bound antibodies and 50S subunit-monomers and larger aggregates by sucrose density gradient centrifugation. The attachment of each of the six immunoglobulins to 50S subunits was visualized directly by electron microscopy using negative staining and correlated with one or more of the structural features of the particle. Each of the proteins was found to occupy a unique position. Proteins L14, L19 and L23 are located in the region of the 50S subunit which interacts with the 30S particle. Proteins L17 and L22 are on the opposite side of the 50S ribosomal subunit, whilst L18 holds a position on one of the lateral protuberances of a crown-like shaped 50S subunit.Two main forms of 50S subunits were seen on the electron micrographs: one of them had a crown-like shape; the second revealed kidney shaped images. It was demonstrated that these two images are only projections of one structure of the subunit that resembled an armchair; each of the forms could be transformed into the other through rotation by 90°.The results allowed the proposal of a three-dimensional model of the 50S subunit with the location of six different ribosomal proteins as illustrated in Fig. 13.

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

SummaryRevertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.

30S Ribosomal proteins associated with the 3′-terminus of 16S RNA

Although it has proven very difficult to develop a generally applicable technique for cross-linking ribosomal proteins to their corresponding RNA binding sites in situ, a very restricted procedure has been available for quite some time. Thus, oxidation of the 3’-terminal ribose of RNA followed by reduction of the product formed with the e-amino group of lysine can create a stable bond between the RNA and an appropriately positioned protein [ 11. Indeed it has been suggested that the ribosomal protein Sl is situated in the intact 30s subunit at a site very near to the 3’-terminus of the 16s RNA [2]. The evidence for this surmise consists of the observation that the proteins extracted from periodate oxidized 30s subunits are deficient in the electrophoretic component corresponding to Sl . Nevertheless no evidence was presented to demonstrate that the missing protein was in fact bound to the 3’-end of the oxidized/reduced intact 16s RNA [2]. Our reexamination of the topographic relationship between 30s protein Sl and the 3’-terminus of the 16s RNA was prompted by recent experiments which suggest a well defined functional relationship between these ribosomal components. Thus, nucleotide sequences at the 3’-end of the 16s RNA are apparently involved in the initiation of protein synthesis [3,4]. Furthermore, the original suggestion that Sl is involved in the binding of mRNA to the ribosome [5]

Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity

An abundantly secreted, highly basic 12-kDa protein (PAF) was purified from the culture medium of Penicillium chrysogenum (Pc). Based on the N-terminal amino acid (aa) sequence of the protein, an oligodeoxyribonucleotide probe was derived and used for amplification of the encoding cDNA by PCR. This cDNA fragment encodes a Cys-rich preproprotein of 92 aa which appears to be processed to a mature product of 55 aa. The deduced aa sequence of the preproprotein reveals 42.6% identity to an antifungal protein (AFP) of Aspergillus giganteus. Agar diffusion tests confirmed that the Pc protein exhibits antifungal activity. In order to investigate the promoter region and the structural organization of the paf gene, a genomic 6-kb fragment was isolated and partially sequenced. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of a coding region of 279 bp which is interrupted by two introns of 76 and 68 bp in length. In the promoter region, a typical TATA box, a motif resembling the fungal carbon catabolite repression element, as well as several putative GATA factor binding motifs, were found. Northern blot analysis indicated that the regulation of paf expression occurs at the level of mRNA transcription and is under control of carbon catabolite and nitrogen metabolite repression regulatory circuits.

Ribosomal proteins. XXV. Immunological studies on Escherichia coli ribosomal proteins.

Abstract Antisera specific for each of the twenty-one 30 s proteins and for 24 50 s ribosomal proteins of Escherichia coli were prepared and used to investigate whether there are any extensive sequence homologies between the different proteins. No immunological cross-reactions were detected between any of the proteins, except L7 and L12 which gave complete cross-reaction. We conclude that the proteins investigated differ substantially in their sequences, but that proteins L7 and L12 have considerable structure homology.

Proteins occurring at, or near, the subunit interface of E. coli ribosomes

SummaryThe identification of ribosomal proteins that occur at, or near, the subunit interface of the 30S and 50S subunits in the E. coli 70S ribosome was attempted by studying the effect of antibodies on the Mg++ dependent dissociation-association equilibrium of 70S ribosomes. Dissociated ribosomes were mixed with monovalent fragments of IgG antibodies (Fabs) specific for each ribosomal protein and then reassociated into intact 70S particles. Various degrees of inhibition of this reassociation were observed for proteins S9, S11, S12, S14, S20, L1, L6, L14, L15, L19, L20, L23, L26 and L27. A small amount of aggregation of 50S subunits was caused by IgGs specific for the proteins S9, S11, S12, S14 and S20 and purified 50S subunits. It was inferred that the presence of small amounts of these proteins on 50S subunits was compatible with their presence at the subunit interface. Finally, the capacity of proteins S11 and S12 to bind to 23S RNA was demonstrated.

Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 S RNA and specific 50 S subunit proteins

Abstract Pancreatic RNase partial digests of 32 P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed.

Effective methods for the investigation of human tear film proteins and lipids

The investigation of human tear film proteins and lipids is of value for the elucidation of contact lens incompatibilities, tear film instabilities, dry eye syndrome and various other eye diseases. Improved efficient methods for the investigation of human tear film proteins and lipids are presented in this paper. Tear proteins were examined by ultrathin sodium dodecyl sulfate-polyacrylamide gel electrophoresis, celluloacetate gel, isoelectric focusing, and high-performance liquid chromatography. The methods differ in sensitivity, resolution, convenience and reproducibility. Tear lipids were examined by high-performance thin-layer chromatography, and good separation into the major lipid classes was achieved. With this method it is possible to examine the lipids present in tears of an individual subject and not just in pools made up from the tears of several persons.