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 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 The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

2004 ◽  
Vol 85 (7) ◽  
pp. 2001-2013 ◽  
Author(s):  
Koen W. R. van Cleef ◽  
Wendy M. A. Scaf ◽  
Karen Maes ◽  
Suzanne J. F. Kaptein ◽  
Erik Beuken ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Replication ◽  
Deletion Mutant ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

scholarly journals Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

1994 ◽  
Vol 127 (6) ◽  
pp. 1617-1626 ◽  
Author(s):  
M Furuse ◽  
M Itoh ◽  
T Hirase ◽  
A Nagafuchi ◽  
S Yonemura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Tight Junctions ◽  
Integral Membrane Protein ◽  
Deletion Mutants ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Vitro Binding ◽  
Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

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 Genetic Analysis, Using P22 Challenge Phage, of the Nitrogen Activator Protein DNA-Binding Site in the Klebsiella aerogenes put Operon

1998 ◽  
Vol 180 (3) ◽  
pp. 571-577 ◽  
Author(s):  
Li-Mei Chen ◽  
Thomas J. Goss ◽  
Robert A. Bender ◽  
Simon Swift ◽  
Stanley Maloy
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Genetic Analysis ◽  
Nitrogen Assimilation ◽  
Regulatory Protein ◽  
Klebsiella Aerogenes ◽  
Dna Binding Site ◽  
Control Protein

ABSTRACT The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons fromKlebsiella aerogenes and Escherichia coli. We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC.

scholarly journals Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

1993 ◽  
Vol 13 (3) ◽  
pp. 1805-1814
Author(s):  
H Wang ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

scholarly journals nSec-1 (munc-18) interacts with both primed and unprimed syntaxin 1A and associates in a dimeric complex on adrenal chromaffin granules

1999 ◽  
Vol 342 (3) ◽  
pp. 707-714 ◽  
Author(s):  
Lee P. HAYNES ◽  
Alan MORGAN ◽  
Robert D. BURGOYNE
Keyword(s):  
Fusion Protein ◽  
Regulatory Protein ◽  
Physical Interaction ◽  
Dimeric Complex ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Attachment Protein ◽  
Syntaxin 1A

The target-SNARE syntaxin 1A is an essential component of the core machinery required for regulated exocytosis (where SNARE is the soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor). Syntaxin 1A interacts with a variety of other proteins, two of which, N-ethylmaleimide-sensitive fusion protein (NSF) and α-soluble NSF attachment protein (α-SNAP) have been suggested to impart a conformational rearrangement on this protein during a reaction referred to as priming. We have studied the effect of the primed state on the binding properties of syntaxin 1A and we have confirmed that primed syntaxin 1A no longer associated with α-SNAP or its cognate vesicle-SNARE, vesicle-associated membrane protein (VAMP). Under such conditions, however, it retained the ability to bind to nSec-1. It has been demonstrated that nSec-1, a regulatory protein also involved in neuronal exocytosis, binds syntaxin 1A with high affinity in vitro, although evidence for this physical interaction occurring in vivo has proven elusive. We analysed the subcellular distribution of these two proteins in fractions from bovine adrenal medulla and detected syntaxin 1A and nSec-1 in both plasma membrane and chromaffin-granule fractions. Using a cross-linking approach with chromaffin-granule membranes we detected a putative dimeric complex composed of approx. 54% total granule membrane nSec-1 and approx. 30% total syntaxin 1A. The results of this study therefore suggest the possibility of nSec-1 interactions with primed syntaxin 1A and demonstrate a potentially significant interaction of syntaxin 1A and nSec-1 on the membranes of chromaffin granules.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

1994 ◽  
Vol 14 (6) ◽  
pp. 3842-3852 ◽  
Author(s):  
C Cheng ◽  
N Kacherovsky ◽  
K M Dombek ◽  
S Camier ◽  
S K Thukral ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Inverted Repeat ◽  
Target Genes ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Potential Target

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.

scholarly journals Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein

2000 ◽  
Vol 20 (9) ◽  
pp. 3049-3057 ◽  
Author(s):  
Daron C. Barnard ◽  
James G. Patton
Keyword(s):  
Regulatory Protein ◽  
Structural Similarity ◽  
Sr Proteins ◽  
Rna Recognition Motif ◽  
Sr Protein ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Rs Domains

ABSTRACT We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

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