scholarly journals Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

1991 ◽  
Vol 11 (4) ◽  
pp. 2169-2179 ◽  
Author(s):  
H F Qui ◽  
E Dubois ◽  
F Messenguy
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Regulatory Protein ◽  
Protein A ◽  
Regulatory Proteins ◽  
Dna Binding Domain ◽  
Zinc Finger Motif ◽  
Activation Domain ◽  
Catabolic Pathways ◽  
Lexa Protein

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways

1991 ◽  
Vol 11 (4) ◽  
pp. 2169-2179
Author(s):  
H F Qui ◽  
E Dubois ◽  
F Messenguy
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Regulatory Protein ◽  
Protein A ◽  
Regulatory Proteins ◽  
Dna Binding Domain ◽  
Zinc Finger Motif ◽  
Activation Domain ◽  
Catabolic Pathways ◽  
Lexa Protein

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis

1988 ◽  
Vol 8 (9) ◽  
pp. 3726-3733
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

scholarly journals In vitro analysis of the zinc-finger motif in human replication protein A

1999 ◽  
Vol 337 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Jiaowang DONG ◽  
Jang-Su PARK ◽  
Suk-Hee LEE
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
Binding Domain ◽  
Replication Protein ◽  
Zinc Finger Motif ◽  
Zinc Finger Domain ◽  
Single Stranded Dna

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase α-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys → Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase δ activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2–3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.

Implications of the zinc‐finger motif found in the DNA‐binding domain of the human XPA protein

1996 ◽  
Vol 1 (5) ◽  
pp. 437-442 ◽  
Author(s):  
Eugene H. Morita ◽  
Tadayasu Ohkubo ◽  
Isao Kuraoka ◽  
Masahiro Shirakawa ◽  
Kiyoji Tanaka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Zinc Finger Motif

Mutational analysis of the GAL4-encoded transcriptional activator protein of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 120 (1) ◽  
pp. 63-74
Author(s):  
M Johnston ◽  
J Dover
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Transcription Activation ◽  
Binding Domain ◽  
Zinc Binding ◽  
Zinc Ions ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcription Activation Domain

Abstract The GAL4 protein of Saccharomyces cerevisiae binds to DNA upstream of each of six genes and stimulates their transcription. To locate regions of the protein responsible for these processes, we identified and characterized 88 gal4 mutations selected in vivo to reduce the ability to GAL4 protein to activate transcription. These mutations alter two regions of GAL4 protein: the DNA binding domain, and the transcription activation domain. Some mutations in the DNA binding domain that abolish the ability of GAL4 protein to bind to DNA in vitro change amino acid residues proposed to form a zinc finger, confirming that this structure is indeed involved in DNA binding. Four different amino acid changes in the zinc finger appear to reduce (but not abolish) the affinity of GAL4 protein for zinc ions, thereby identifying some of the amino acids involved in forming the zinc-binding structure. Several other mutations that abolish the DNA binding activity of the protein alter the 20 amino acids adjacent to the zinc finger, suggesting that these residues are part of the DNA binding domain. Two amino acid changes in the region adjacent to the zinc finger also appear to affect the ability of GAL4 protein to bind zinc ions, suggesting that this region of the protein can influence the structure of the zinc binding domain. The transcription activation domain of GAL4 protein is remarkably resistant to single amino acid changes: only 4 of the 42 mutations that alter this region of the protein are of the missense type. This observation is consistent with other lines of evidence that GAL4 protein possesses multiple transcription activation domains with unusual sequence flexibility.

scholarly journals Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

1988 ◽  
Vol 8 (9) ◽  
pp. 3726-3733 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Activity ◽  
Carboxyl Terminal

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

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