1,720,975 research outputs found
Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae
No full textControl of transcription and enzyme activities are two interwoven regulatory systems essential for the function of a metabolic node. Saccharomyces cerevisiae strains differing in enzyme activities at the chorismate branch point of aromatic amino acid biosynthesis were constructed by recombinant DNA technology. Expression of an allosterically unregulated, constitutively activated chorismate mutase encoded by the ARO7T(226I) (ARO7(c)) allele depleted the chorismate pool. The resulting tryptophan limitation caused growth defects, which could be counteracted only by transcriptional induction of TRP2 encoding the competing enzyme anthranilate synthase. ARO7 expression is not transcriptionally regulated by amino acids. Transcriptional activation of the ARO7(c) allele led to stronger growth retardation upon tryptophan limitation. The same effect was achieved by removing the competing enzyme anthranilate synthase, which is encoded by the TRP2 gene, from the transcriptional control. The allelic situation of ARO7(c) being under general control instead of TRP2 resulted in severe growth defects when cells were starved for tryptophan. In conclusion, the specific regulatory pattern acting on enzymatic activities at the first metabolic node of aromatic amino acid biosynthesis is necessary to maintain proper flux distribution. Therefore, the evolution of the sophisticated allosteric regulation of yeast chorismate mutase requires as prerequisite (i) that the encoding ARO7 gene is not transcriptionally regulated, whereas (ii) the transcription of the competing feedback-regulated anthranilate synthase-encoding gene is controlled by availability of amino acids.NIGMS NIH HHS [R01 GM006920
Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase
No full textThe yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.NIGMS NIH HHS [R01 GM006920, GM06920
Evolution of feedback-inhibited beta/alpha barrel isoenzymes by gene duplication and a single mutation
No full textThe beta/alpha barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deOXY-D-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the beta/alpha barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.NIGMS NIH HHS [R01 GM006920
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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