scholarly journals Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding.

1992 ◽  
Vol 12 (5) ◽  
pp. 1940-1949 ◽  
Author(s):  
A D Keller ◽  
T Maniatis
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Specific Binding ◽  
Regulatory Element ◽  
Zinc Fingers ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Binding Activity ◽  
Necessary And Sufficient ◽  
Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

1992 ◽  
Vol 12 (5) ◽  
pp. 1940-1949
Author(s):  
A D Keller ◽  
T Maniatis
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Specific Binding ◽  
Regulatory Element ◽  
Zinc Fingers ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Binding Activity ◽  
Necessary And Sufficient ◽  
Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

scholarly journals An unusual arrangement of 13 zinc fingers in the vertebrate gene Z13

1995 ◽  
Vol 311 (1) ◽  
pp. 219-224 ◽  
Author(s):  
T C Schulz ◽  
B Hopwood ◽  
P D Rathjen ◽  
J R Wells
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Structural Organization ◽  
Zinc Fingers ◽  
Binding Activity ◽  
Protein Domain ◽  
Nucleic Acid Binding ◽  
C Terminus ◽  
Wide Range

The zinc finger is a protein domain that imparts specific nucleic acid-binding activity on a wide range of functionally important proteins. In this paper we report the molecular cloning and characterization of a novel murine zinc-finger gene, mZ13. Analysis of mZ13 cDNAs revealed that the gene expresses a 794-amino-acid protein encoded by a 2.7 kb transcript. The protein has an unusual arrangement of 13 zinc fingers into a ‘hand’ of 12 tandem fingers and a single isolated finger near the C-terminus. This structural organization is conserved with the probable chicken homologue, cZ13. mZ13 also contained an additional domain at the N-terminus which has previously been implicated in the regulation of zinc-finger transcription factor DNA-binding, via protein-protein interactions. mZ13 expression was detected in a wide range of murine embryonic and adult tissues. The structural organization of mZ13 and its expression profile suggest that it may function as a housekeeping DNA-binding protein that regulates the expression of specific genes.

A maternal factor, OZ-1, activates embryonic transcription of the Xenopus laevis GS17 gene

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 649-655 ◽  
Author(s):  
N. Ovsenek ◽  
A.M. Zorn ◽  
P.A. Krieg
Keyword(s):  
Dna Binding ◽  
Xenopus Laevis ◽  
Specific Binding ◽  
Regulatory Element ◽  
Binding Activity ◽  
Developmental Expression ◽  
Promoter Regions ◽  
Enhancer Activity ◽  
Sequence Element ◽  
Binding Factor

We describe the identification of an enhancer sequence and a sequence-specific DNA-binding protein required for developmental expression of the Xenopus laevis GS17 gene. Using microinjection of recombinant plasmids into fertilized frog eggs, we have shown that a 14 base pair CT-rich sequence element, normally located about 700 bases upstream of the GS17 promoter, is sufficient to activate transcription of a heterologous reporter gene in gastrula stage embryos. This regulatory element has been called the OZ sequence. Sequences closely related to OZ are located in the promoter regions of several other genes expressed during Xenopus development. Extracts prepared from Xenopus embryos show the presence of a DNA-binding factor, OZ-1, that specifically recognizes the OZ sequence. Mutations within the OZ element that abolish OZ-1 binding also abolish enhancer activity. The OZ-1 factor contains at least two proteins of approximate M(r) 76 × 10(3) and 100 × 10(3). The sequence-specific binding activity accumulates during oogenesis and remains present at approximately constant levels throughout early development.

scholarly journals Inducible processing of interferon regulatory factor-2.

1992 ◽  
Vol 12 (8) ◽  
pp. 3325-3336 ◽  
Author(s):  
V J Palombella ◽  
T Maniatis
Keyword(s):  
Amino Acids ◽  
Regulatory Element ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Interferon Regulatory Factor ◽  
Binding Activity ◽  
Regulatory Factor ◽  
Truncated Form ◽  
Terminal Amino ◽  
Beta Gene

PRDI-BFc and PRDI-BFi are proteins that bind specifically to a regulatory element required for virus induction of the human beta interferon (IFN-beta). PRDI-BFc is a constitutive binding activity, while the PRDI-BFi binding activity is observed only after cells are treated with inducers such as virus or poly(I).poly(C) plus cycloheximide or in some cells by cycloheximide alone. In this paper we report that PRDI-BFc is interferon regulatory factor-2 (IRF-2), a known transcriptional repressor. In addition, we find that PRDI-BFi is a truncated form of IRF-2, lacking approximately 185 C-terminal amino acids. Thus, PRDI-BFi appears to be generated by inducible proteolysis. Although the affinity of PRDI-BFc/IRF-2 for the IFN-beta promoter does not appear to be affected by the removal of C-terminal amino acids, the ability of PRDI-BFi to function as a repressor in cotransfection experiments is significantly less than that of intact IRF-2. Studies have shown that IRF-2 can block the activity of the transcriptional activator IRF-1, which also binds specifically to the IFN-beta gene promoter. Thus, the inducible proteolysis of IRF-2 may be involved in the regulation of the IFN-beta gene or of other genes in which the ratio of IRF-1 to IRF-2 can affect the level of transcription.

scholarly journals The Zinc-Sensing Mechanism of Mouse MTF-1 Involves Linker Peptides between the Zinc Fingers

2006 ◽  
Vol 26 (15) ◽  
pp. 5580-5587 ◽  
Author(s):  
Yong Li ◽  
Tomoki Kimura ◽  
John H. Laity ◽  
Glen K. Andrews
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Zinc Fingers ◽  
Gene Promoter ◽  
Zinc Sensing ◽  
Cellular Zinc

ABSTRACT Mouse metal response element-binding transcription factor-1 (MTF-1) regulates the transcription of genes in response to a variety of stimuli, including exposure to zinc or cadmium, hypoxia, and oxidative stress. Each of these stresses may increase labile cellular zinc, leading to nuclear translocation, DNA binding, and transcriptional activation of metallothionein genes (MT genes) by MTF-1. Several lines of evidence suggest that the highly conserved six-zinc finger DNA-binding domain of MTF-1 also functions as a zinc-sensing domain. In this study, we investigated the potential role of the peptide linkers connecting the four N-terminal zinc fingers of MTF-1 in their zinc-sensing function. Each of these three linkers is unique, completely conserved among all known vertebrate MTF-1 orthologs, and different from the canonical Cys2His2 zinc finger TGEKP linker sequence. Replacing the RGEYT linker between zinc fingers 1 and 2 with TGEKP abolished the zinc-sensing function of MTF-1, resulting in constitutive DNA binding, nuclear translocation, and transcriptional activation of the MT-I gene. In contrast, swapping the TKEKP linker between fingers 2 and 3 with TGEKP had little effect on the metal-sensing functions of MTF-1, whereas swapping the canonical linker for the shorter TGKT linker between fingers 3 and 4 rendered MTF-1 less sensitive to zinc-dependent activation both in vivo and in vitro. These observations suggest a mechanism by which physiological concentrations of accessible cellular zinc affect MTF-1 activity. Zinc may modulate highly specific, linker-mediated zinc finger interactions in MTF-1, thus affecting its zinc- and DNA-binding activities, resulting in translocation to the nucleus and binding to the MT-I gene promoter.

scholarly journals Inducible processing of interferon regulatory factor-2

1992 ◽  
Vol 12 (8) ◽  
pp. 3325-3336
Author(s):  
V J Palombella ◽  
T Maniatis
Keyword(s):  
Amino Acids ◽  
Regulatory Element ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Interferon Regulatory Factor ◽  
Binding Activity ◽  
Regulatory Factor ◽  
Truncated Form ◽  
Terminal Amino ◽  
Beta Gene

PRDI-BFc and PRDI-BFi are proteins that bind specifically to a regulatory element required for virus induction of the human beta interferon (IFN-beta). PRDI-BFc is a constitutive binding activity, while the PRDI-BFi binding activity is observed only after cells are treated with inducers such as virus or poly(I).poly(C) plus cycloheximide or in some cells by cycloheximide alone. In this paper we report that PRDI-BFc is interferon regulatory factor-2 (IRF-2), a known transcriptional repressor. In addition, we find that PRDI-BFi is a truncated form of IRF-2, lacking approximately 185 C-terminal amino acids. Thus, PRDI-BFi appears to be generated by inducible proteolysis. Although the affinity of PRDI-BFc/IRF-2 for the IFN-beta promoter does not appear to be affected by the removal of C-terminal amino acids, the ability of PRDI-BFi to function as a repressor in cotransfection experiments is significantly less than that of intact IRF-2. Studies have shown that IRF-2 can block the activity of the transcriptional activator IRF-1, which also binds specifically to the IFN-beta gene promoter. Thus, the inducible proteolysis of IRF-2 may be involved in the regulation of the IFN-beta gene or of other genes in which the ratio of IRF-1 to IRF-2 can affect the level of transcription.

scholarly journals Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

2016 ◽  
Vol 72 (9) ◽  
pp. 713-719 ◽  
Author(s):  
Qiujia Chen ◽  
Millie Georgiadis
Keyword(s):  
Dna Binding ◽  
Catalytic Domain ◽  
Specific Binding ◽  
Critical Role ◽  
Binding Activity ◽  
Structural Basis ◽  
Unexpected Finding ◽  
Terminal Inverted Repeat ◽  
Dna Binding Activity ◽  
New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

scholarly journals The Human Monocytic Leukemia Zinc Finger Histone Acetyltransferase Domain Contains DNA-binding Activity Implicated in Chromatin Targeting

2007 ◽  
Vol 282 (50) ◽  
pp. 36603-36613 ◽  
Author(s):  
Marc A. Holbert ◽  
Timothy Sikorski ◽  
Juliana Carten ◽  
Danielle Snowflack ◽  
Santosh Hodawadekar ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Histone Acetyltransferase ◽  
Binding Activity ◽  
Monocytic Leukemia ◽  
Dna Binding Activity ◽  
Human Monocytic Leukemia ◽  
Acetyltransferase Domain

Characterization of the DNA Binding Activity of the ZFY Zinc Finger Domain

Biochemistry ◽  
2010 ◽  
Vol 49 (4) ◽  
pp. 679-686 ◽  
Author(s):  
Jennifer Grants ◽  
Erin Flanagan ◽  
Andrea Yee ◽  
Paul J. Romaniuk
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Binding Activity ◽  
Zinc Finger Domain ◽  
Dna Binding Activity

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

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