scholarly journals Mutational Analysis of the Tup1 General Repressor of Yeast

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 637-644
Author(s):  
Pauline M Carrico ◽  
Richard S Zitomer
Keyword(s):  
Mating Type ◽  
Transcriptional Repression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Repeat Region ◽  
Identify Amino Acid ◽  
Repression Complex ◽  
Mating Type Genes

Abstract The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.

scholarly journals Mutational Analysis of Xanthomonas Harpin HpaG Identifies a Key Functional Region That Elicits the Hypersensitive Response in Nonhost Plants

2004 ◽  
Vol 186 (18) ◽  
pp. 6239-6247 ◽  
Author(s):  
Jung-Gun Kim ◽  
Eunkyung Jeon ◽  
Jonghee Oh ◽  
Jae Sun Moon ◽  
Ingyu Hwang
Keyword(s):  
Amino Acid ◽  
Hypersensitive Response ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Tobacco Plants ◽  
Functional Region ◽  
Amino Acid Peptide ◽  
Α Helix

ABSTRACT HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an α-helix, as is the HrpW region that is homologous to HpaG. The predicted α-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.

scholarly journals Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Lina Kaminski ◽  
Jerry Eichler
Keyword(s):  
Biochemical Characterization ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Natural Substrate ◽  
Identify Amino Acid ◽  
Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

scholarly journals Mutational Analysis of the Caenorhabditis elegans Cell-Death Gene ced-3

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1655-1671
Author(s):  
Shai Shaham ◽  
Peter W Reddien ◽  
Brian Davies ◽  
H Robert Horvitz
Keyword(s):  
Cell Death ◽  
Caenorhabditis Elegans ◽  
Programmed Cell Death ◽  
Mutational Analysis ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Active Site Cysteine ◽  
Related Proteins

Abstract Mutations in the gene ced-3, which encodes a protease similar to interleukin-1β converting enzyme and related proteins termed caspases, prevent programmed cell death in the nematode Caenorhabditis elegans. We used site-directed mutagenesis to demonstrate that both the presumptive active-site cysteine of the CED-3 protease and the aspartate residues at sites of processing of the CED-3 proprotein are required for programmed cell death in vivo. We characterized the phenotypes caused by and the molecular lesions of 52 ced-3 alleles. These alleles can be ordered in a graded phenotypic series. Of the 30 amino acid sites altered by ced-3 missense mutations, 29 are conserved with at least one other caspase, suggesting that these residues define sites important for the functions of all caspases. Animals homozygous for the ced-3(n2452) allele, which is deleted for the region of the ced-3 gene that encodes the protease domain, seemed to be incompletely blocked in programmed cell death, suggesting that some programmed cell death can occur independently of CED-3 protease activity.

scholarly journals Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase

1998 ◽  
Vol 180 (17) ◽  
pp. 4613-4620 ◽  
Author(s):  
Takayuki Horiuchi ◽  
Teruya Komano
Keyword(s):  
Mutational Analysis ◽  
Minor Component ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Missense Mutations ◽  
Class Iii ◽  
Mating Activity ◽  
Type Iv ◽  
Prepilin Peptidase ◽  
Plasmid R64

ABSTRACT The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. ThepilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of thepilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with thepilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.

scholarly journals Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

1993 ◽  
Vol 177 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Y Lobet ◽  
C Feron ◽  
G Dequesne ◽  
E Simoen ◽  
P Hauser ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Pertussis Toxin ◽  
Neutralizing Antibodies ◽  
Whooping Cough ◽  
Site Directed Mutagenesis ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
Generation Vaccine ◽  
Adp Ribosyltransferase

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

scholarly journals Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii

1999 ◽  
Vol 344 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Jae Hyung AN ◽  
Gha Young LEE ◽  
Jin-Won JUNG ◽  
Weontae LEE ◽  
Yu Sam KIM
Keyword(s):  
Enzyme Catalysis ◽  
Hplc Analysis ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Database Analysis ◽  
Identify Amino Acid ◽  
Malonyl Coa

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either.

MGDG, PG and SQDG regulate the activity of light-dependent protochlorophyllide oxidoreductase

2017 ◽  
Vol 474 (7) ◽  
pp. 1307-1320 ◽  
Author(s):  
Michal Gabruk ◽  
Beata Mysliwa-Kurdziel ◽  
Jerzy Kruk
Keyword(s):  
Molecular Mechanisms ◽  
Chlorophyll Biosynthesis ◽  
Fluorescence Emission ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Biosynthesis Pathway ◽  
Amino Acid Residues ◽  
Protochlorophyllide Oxidoreductase ◽  
Emission Maximum ◽  
Identify Amino Acid

Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654 nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV

Biochemistry ◽  
1992 ◽  
Vol 31 (20) ◽  
pp. 4808-4815 ◽  
Author(s):  
Ursel Selent ◽  
Thomas Rueter ◽  
Eleonore Koehler ◽  
Michaela Liedtke ◽  
Vera Thielking ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Endonuclease ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Identify Amino Acid ◽  
A Site ◽  
Mutagenesis Study

scholarly journals Mutational analysis of Rox1, a DNA-bending repressor of hypoxic genes in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (11) ◽  
pp. 6109-6117 ◽  
Author(s):  
J Deckert ◽  
A M Rodriguez Torres ◽  
J T Simon ◽  
R S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Structural Model ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Upstream Region ◽  
Missense Mutations ◽  
Bent Dna ◽  
Repression Complex ◽  
Carboxy Terminal

Rox1 is a repressor of the hypoxic genes of Saccharomyces cerevisiae. It binds to a specific hypoxic consensus sequence in the upstream region of these genes and represses transcription in conjunction with the general repression complex Tup1-Ssn6. In this study, we demonstrated that the first 100 amino acids comprising the HMG domain of Rox1 were responsible for DNA binding and that when bound, Rox1 bent DNA at an angle of 90 degrees. A mutational analysis resulted in the isolation of seven missense mutations, all located within the HMG domain, that caused loss of DNA binding. The effect of these mutations on the structure of Rox1 was evaluated on the basis of the homology between Rox1 and the human male sex-determining protein SRY, for which a structural model is available. The failure to isolate missense mutations in the carboxy-terminal three-quarters of the protein prompted a deletion analysis of this region. The results suggested that this region was responsible for the repression function of Rox1 and that the repression information was redundant. This hypothesis was confirmed by using a set of fusions between sequences encoding the GAL4 DNA-binding domain and portions of ROX1. Those fusions containing either the entire carboxy-terminal region or either half of it were capable of repression. Repression by selected fusions was demonstrated to be dependent on Ssn6.

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