scholarly journals Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding

2000 ◽  
Vol 182 (20) ◽  
pp. 5807-5812 ◽  
Author(s):  
Eun Hee Cho ◽  
Renato Alcaraz ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Glutamic Acid ◽  
Cooperative Interaction ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Factor For Inversion Stimulation ◽  
Α Helix

ABSTRACT The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.

scholarly journals Assembly of amino-terminally deleted desmin in vimentin-free cells.

1990 ◽  
Vol 111 (5) ◽  
pp. 1971-1985 ◽  
Author(s):  
J M Raats ◽  
F R Pieper ◽  
W T Vree Egberts ◽  
K N Verrijp ◽  
F C Ramaekers ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hela Cells ◽  
Filament Formation ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Double Label ◽  
Amino Terminal Domain ◽  
Mcf 7 ◽  
Vimentin Filaments

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

scholarly journals Mutational Analysis of the MutH Protein fromEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 12113-12119 ◽  
Author(s):  
Tamalette Loh ◽  
Kenan C. Murphy ◽  
Martin G. Marinus
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Flexible Loop ◽  
Loop Regions ◽  
Binding Residues

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH andPvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis asPvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops inPvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) inPvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity bothin vivoandin vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

scholarly journals Characterization of MarR Superrepressor Mutants

1999 ◽  
Vol 181 (10) ◽  
pp. 3303-3306 ◽  
Author(s):  
Michael N. Alekshun ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Antibiotic Resistance ◽  
Dna Binding ◽  
Multiple Antibiotic Resistance ◽  
Mutant Proteins ◽  
Whole Cells

ABSTRACT MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.

scholarly journals Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369 ◽  
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1

1989 ◽  
Vol 9 (6) ◽  
pp. 2360-2369
Author(s):  
S K Thukral ◽  
M A Tavianini ◽  
H Blumberg ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Deletion Mutant ◽  
Regulatory Protein ◽  
Deletion Mutants ◽  
Amino Terminal ◽  
Beta Galactosidase

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

scholarly journals Iron Activates In Vivo DNA Binding of Schizosaccharomyces pombe Transcription Factor Fep1 through Its Amino-Terminal Region

2009 ◽  
Vol 8 (4) ◽  
pp. 649-664 ◽  
Author(s):  
Mehdi Jbel ◽  
Alexandre Mercier ◽  
Benoit Pelletier ◽  
Jude Beaudoin ◽  
Simon Labbé
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Schizosaccharomyces Pombe ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Fluorescent Protein ◽  
Chromatin Binding ◽  
Iron Starvation ◽  
Amino Terminal

ABSTRACT In Schizosaccharomyces pombe, the iron sensor Fep1 mediates the transcriptional repression of iron transport genes in response to high concentrations of iron. On the other hand, fep1 + expression is downregulated under conditions of iron starvation by the CCAAT-binding factor Php4. In this study, we created a fep1Δ php4Δ double mutant strain where expression of fep1 + was disengaged from its iron limitation-dependent repression by Php4 to examine the effects of iron on constitutively expressed functional fep1 + -GFP and TAP-fep1 + alleles and their gene products. In these cells, Fep1-green fluorescent protein was invariably localized in the nucleus under both iron-limiting and iron-replete conditions. Using chromatin immunoprecipitation assays, we found that Fep1 is associated with iron-responsive promoters in vivo. Chromatin binding was iron dependent, with a loss of binding observed in the presence of low iron. Functional dissection of the protein revealed that the N-terminal 241-residue segment that includes two consensus Cys2/Cys2-type zinc finger motifs and a Cys-rich region is required for optimal promoter occupancy by Fep1. Within this segment, a minimal module encompassing amino acids 60 to 241 is sufficient for iron-dependent chromatin binding. Using yeast one-hybrid analysis, we showed that the replacement of the repression domain of Fep1 by fusing the activation domain of VP16 to the chromatin-binding fragment of amino acids 1 to 241 of Fep1 converts the protein from an iron-dependent repressor into an iron-dependent transcriptional activator. Thus, the repression function of Fep1 can be replaced with that of a transcriptional activation function without the loss of its iron-dependent DNA-binding activity.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 29-35
Author(s):  
D N Arvidson ◽  
M Shapiro ◽  
P Youderian
Keyword(s):  
Dna Binding ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Mutant Proteins ◽  
Naturally Occurring ◽  
Repressor Complex ◽  
Genes Encoding ◽  
Active Repressor

Abstract The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

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