scholarly journals The N Terminus of the Centromere H3-Like Protein Cse4p Performs an Essential Function Distinct from That of the Histone Fold Domain

2000 ◽  
Vol 20 (18) ◽  
pp. 7037-7048 ◽  
Author(s):  
Yinhuai Chen ◽  
Richard E. Baker ◽  
Kevin C. Keith ◽  
Kendra Harris ◽  
Sam Stoler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Proteins ◽  
Histone Fold ◽  
Human Centromere ◽  
Kinetochore Assembly ◽  
Evolutionarily Conserved ◽  
Histone Fold Domain ◽  
N Terminus ◽  
Acid Domain

ABSTRACT Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation.

scholarly journals The Histone Fold Domain of Cse4 Is Sufficient for CEN Targeting and Propagation of Active Centromeres in Budding Yeast

2004 ◽  
Vol 3 (6) ◽  
pp. 1533-1543 ◽  
Author(s):  
Lisa Morey ◽  
Kelly Barnes ◽  
Yinhuai Chen ◽  
Molly Fitzgerald-Hayes ◽  
Richard E. Baker
Keyword(s):  
Budding Yeast ◽  
Fold Increase ◽  
Chromosome Loss ◽  
Wild Type ◽  
Mutant Proteins ◽  
Histone Fold ◽  
Kinetochore Assembly ◽  
Histone Fold Domain ◽  
Terminal Amino

ABSTRACT Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly. Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae. Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT). The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal. To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4ΔNT) were analyzed. No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4. A Cse4 degron strain was used to examine targeting of a Cse4ΔNT protein in the absence of wild-type Cse4. The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting. Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S. cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Δ129) were constructed. Despite the complete absence of the NT, mitotic chromosome loss in the cse4Δ129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4. Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S. cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast.

scholarly journals Mapping and Functional Characterization of the TAF11 Interaction with TFIIA

2005 ◽  
Vol 25 (3) ◽  
pp. 945-957 ◽  
Author(s):  
M. M. Robinson ◽  
G. Yatherajam ◽  
R. T. Ranallo ◽  
A. Bric ◽  
M. R. Paule ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Regulation Of Gene Expression ◽  
Small Subunit ◽  
Molecular Organization ◽  
Functional Link ◽  
Essential Information ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Promoter Dna

ABSTRACT TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

scholarly journals The TFIID Components Human TAFII140 andDrosophila BIP2 (TAFII155) Are Novel Metazoan Homologues of Yeast TAFII47 Containing a Histone Fold and a PHD Finger

2001 ◽  
Vol 21 (15) ◽  
pp. 5109-5121 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Jean-Christophe Pointud ◽  
Sylvie Thuault ◽  
Lucie Carré ◽  
Christophe Romier ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Protein A ◽  
Amino Acid Sequences ◽  
Molecular Organization ◽  
Phd Finger ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
High Degree

ABSTRACT The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAFIIs). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAFIIs and overall molecular organization. In recent years, it has been assumed that all the metazoan TAFIIs have been identified, yet no metazoan homologues of yeast TAFII47 (yTAFII47) and yTAFII65 are known. Both of these yTAFIIs contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAFII25. We have cloned a novel mouse protein, TAFII140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAFII140 shows extensive sequence similarity toDrosophila BIP2 (dBIP2) (dTAFII155), which we also show to be a component of DrosophilaTFIID. These proteins are metazoan homologues of yTAFII47 as their HFDs selectively heterodimerize with dTAFII24 and human TAFII30, metazoan homologues of yTAFII25. We further show that yTAFII65 shares two domains with theDrosophila Prodos protein, a recently described potential dTAFII. These conserved domains are critical for yTAFII65 function in vivo. Our results therefore identify metazoan homologues of yTAFII47 and yTAFII65.

scholarly journals Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ

1999 ◽  
Vol 181 (24) ◽  
pp. 7531-7544 ◽  
Author(s):  
Xiaolan Ma ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Immunoblot Analysis ◽  
Conserved Residues ◽  
Core Domain ◽  
Mutant Proteins ◽  
E Coli ◽  
Stable Mutant ◽  
Functional Analyses

ABSTRACT In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coliFtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomalftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

scholarly journals A proline repeat domain in the Notch co-activator MAML1 is important for the p300-mediated acetylation of MAML1

2007 ◽  
Vol 404 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Mariana Saint Just Ribeiro ◽  
Magnus L. Hansson ◽  
Annika E. Wallberg
Keyword(s):  
Target Genes ◽  
Repeat Motif ◽  
Terminal Domain ◽  
Evolutionarily Conserved ◽  
Repeat Domain ◽  
Intracellular Receptor ◽  
N Terminus ◽  
Lysine Residues ◽  
Receptor Domain

Ligand activation of Notch leads to the release of Notch IC (the intracellular receptor domain), which translocates to the nucleus and interacts with the DNA-binding protein CSL to control expression of specific target genes. In addition to ligand-mediated activation, Notch signalling can be further modulated by interactions of Notch IC with a number of other proteins. MAML1 has previously been shown to act co-operatively with the histone acetyltransferase p300 in Notch IC-mediated transcription. In the present study we show that the N-terminal domain of MAML1 directly interacts with both p300 and histones, and the p300–MAML1 complex specifically acetylates histone H3 and H4 tails in chromatin. Furthermore, p300 acetylates MAML1 and evolutionarily conserved lysine residues in the MAML1 N-terminus are direct substrates for p300-mediated acetylation. The N-terminal domain of MAML1 contains a proline repeat motif (PXPAAPAP) that was previously shown to be present in p53 and important for the p300–p53 interaction. We show that the MAML1 proline repeat motif interacts with p300 and enhances the activity of the MAML1 N-terminus in vivo. These findings suggest that the N-terminal domain of MAML1 plays an important role in Notch-regulated transcription, by direct interactions with Notch, p300 and histones.

scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

scholarly journals Histone Folds Mediate Selective Heterodimerization of Yeast TAFII25 with TFIID Components yTAFII47 and yTAFII65 and with SAGA Component ySPT7

2001 ◽  
Vol 21 (5) ◽  
pp. 1841-1853 ◽  
Author(s):  
Yann-Gaël Gangloff ◽  
Steven L. Sanders ◽  
Christophe Romier ◽  
Doris Kirschner ◽  
P. Anthony Weil ◽  
...  
Keyword(s):  
Temperature Sensitive ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Conserved Residues ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Necessary And Sufficient ◽  
Two Hybrid ◽  
Temperature Sensitive Phenotype

ABSTRACT We show that the yeast TFIID (yTFIID) component yTAFII47 contains a histone fold domain (HFD) with homology to that previously described for hTAFII135. Complementation in vivo indicates that the yTAFII47 HFD is necessary and sufficient for vegetative growth. Mutation of highly conserved residues in the α1 helix of the yTAFII47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAFII25, as well as by yTAFII40, yTAFII19, and yTAFII60. In yeast two-hybrid and bacterial coexpression assays, the yTAFII47 HFD selectively heterodimerizes with yTAFII25, which we show contains an HFD with homology to the hTAFII28 family We additionally demonstrate that yTAFII65 contains a functional HFD which also selectively heterodimerizes with yTAFII25. These results reveal the existence of two novel histone-like pairs in yTFIID. The physical and genetic interactions described here show that the histone-like yTAFIIs are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested. Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAFII47 which selectively heterodimerizes with yTAFII25, defining a novel histone-like pair in the SAGA complex.

scholarly journals Saccharomyces cerevisiae BUR6 encodes a DRAP1/NC2alpha homolog that has both positive and negative roles in transcription in vivo.

1997 ◽  
Vol 17 (4) ◽  
pp. 2057-2065 ◽  
Author(s):  
G Prelich
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Nucleotide Sequence Analysis ◽  
Detectable Effect ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Tata Box Binding Protein ◽  
Mutant Phenotypes ◽  
Carboxy Terminal

BUR3 and BUR6 were identified previously by selecting for mutations that increase transcription from an upstream activating sequence (UAS)-less promoter in Saccharomyces cerevisiae. The bur3-1 and bur6-1 mutations are recessive, increase transcription from a suc2 delta uas allele, and cause other mutant phenotypes, suggesting that Bur3p and Bur6p function as general repressors of the basal transcriptional machinery. The molecular cloning and characterization of BUR3 and BUR6 are presented here. BUR3 is identical to MOT1, a previously characterized essential gene that encodes an ATP-dependent inhibitor of the TATA box-binding protein. Cloning and nucleotide sequence analysis reveals that BUR6 encodes a homolog of DRAP1 (also called NC2alpha), a mammalian repressor of basal transcription. Strains that contain a bur6 null allele are viable but grow extremely poorly, demonstrating that BUR6 is critical for normal cell growth in yeast. The Bur6p histone fold domain is required for function; an extensive nonoverlapping set of deletion alleles throughout the histone fold domain impairs BUR6 function in vivo, whereas mutations in the amino- and carboxy-terminal tails have no detectable effect. BUR6 and BUR3/MOT1 have different functions depending on promoter context: although the bur3-1 and bur6-1 mutations increase transcription from delta uas promoters, they result in reduced transcription from the wild-type GAL1 and GAL10 promoters. This transcriptional defect is due to the inability of the GAL10 UAS to function in bur6-1 strains. The similar phenotypes of bur6 and bur3 (mot1) mutations suggest that Bur6p and Mot1p have related, but not identical, functions in modulating the activity of the general transcription machinery in vivo.

scholarly journals A Molecular Genetic Dissection of the Evolutionarily Conserved N Terminus of Yeast Rad52

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 549-562
Author(s):  
Uffe H Mortensen ◽  
Naz Erdeniz ◽  
Qi Feng ◽  
Rodney Rothstein
Keyword(s):  
Dna Damage ◽  
Amino Acid ◽  
Dna Binding ◽  
Molecular Genetic ◽  
Mitotic Recombination ◽  
Amino Acid Residues ◽  
Γ Irradiation ◽  
Evolutionarily Conserved ◽  
N Terminus ◽  
Γ Ray

Abstract Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of γ-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient γ-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair γ-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of γ-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by γ-irradiation.

scholarly journals The Plant NF-Y DNA Matrix In Vitro and In Vivo

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 406 ◽  
Author(s):  
Nerina Gnesutta ◽  
Matteo Chiara ◽  
Andrea Bernardini ◽  
Matteo Balestra ◽  
David S. Horner ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Sequence Specificity ◽  
Histone Fold ◽  
Nuclear Factor Y ◽  
Histone Fold Domain ◽  
Genes Encoding ◽  
Core Histones ◽  
Ccaat Box

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

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