ALBERT — All Library Books, journals and Electronic Records Telegrafenberg

1

Electronic Resource

Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine (1995)

Stein, Torsten ; Kluge, Britta ; Vater, Joachim ; [et al.]

s.l. : American Chemical Society

Biochemistry 34 (1995), S. 4633-4642

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ISSN: 1520-4995

Source: ACS Legacy Archives

Topics: Biology , Chemistry and Pharmacology

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1021/bi00014a017

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2

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A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes (1996)

Lisowsky, Thomas ; Stein, Torsten ; Michaelis, Georg ; [et al.]

Springer

Current genetics 30 (1996), S. 389-395

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ISSN: 1432-0983

Keywords: Key words Mitochondrial transcription apparatus ; Mitochondrial RNA polymerases ; Nuclear pet mutations ; Saccharomyces cerevisisae

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002940050147

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3

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Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae (1998)

Stein, Torsten ; Kricke, Jörn ; Becher, Dietmar ; [et al.]

Springer

Current genetics 34 (1998), S. 287-296

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ISSN: 1432-0983

Keywords: Key words Zinc-finger protein ; Nuclear localization ; Immuno electron microscopy ; Saccharomyces cerevisiae

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002940050398

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4

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Expression studies and promoter analysis of the nuclear gene for mitochondrial transcription factor 1 (MTF1) in yeast (1999)

Jan, Pey-Shynan ; Stein, Torsten ; Hehl, Stanetta ; [et al.]

Springer

Current genetics 36 (1999), S. 37-48

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ISSN: 1432-0983

Keywords: Key words Yeast ; Mitochondrial transcription ; Specificity factor MTF1 ; Heterologous gene expression ; Regulation

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5′ regions from S. douglasii, S. kluyveri and Kluyveromyces lactis were determined. A comparison of these sequences identifies significant differences and rapid changes in the intergenic regions, even between closely related yeast species.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002940050470

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5

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Bacillus subtilis antibiotics: structures, syntheses and specific functions (2005)

Stein, Torsten

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 56 (2005), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The endospore-forming rhizobacterium Bacillus subtilis– the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the ‘pure’ anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a ‘killing factor’ effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04587.x

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6

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Dual control of subtilin biosynthesis and immunity in Bacillus subtilis (2002)

Stein, Torsten ; Borchert, Stefan ; Kiesau, Peter ; [et al.]

Oxford, UK : Blackwell Science Ltd.

Molecular microbiology 44 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and β-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. β-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02869.x

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7

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The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase (2002)

Pisa, René ; Stein, Torsten ; Eichler, Robert ; [et al.]

Oxford, UK : Blackwell Science Ltd.

Molecular microbiology 43 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenesΔnrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.02784.x

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8

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The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis (2003)

Stein, Torsten ; Heinzmann, Stefan ; Kiesau, Peter ; [et al.]

Oxford, UK : Blackwell Science, Ltd

Molecular microbiology 47 (2003), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03374.x

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9

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The Modular Organization of Multifunctional Peptide Synthetases (1997)

Vater, Joachim ; Stein, Torsten ; Vollenbroich, Dirk ; [et al.]

Springer

The protein journal 16 (1997), S. 557-564

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ISSN: 1573-4943

Keywords: Peptide synthetases ; modular structure ; thioester binding site ; multiple 4′-phosphopantetheine cofactors ; electrospray mass spectrometry ; active-site mutagenesis

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme–inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4′-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the “Multiple Carrier Model” of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1026386100259

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