Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.

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Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.123-132.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 123-132

Author(s):

A D Sharrocks ◽

H Gille ◽

P E Shaw

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Serum Response Factor ◽

Alpha Helix ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Amino Acid Peptide ◽

Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

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A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300197 ◽

2003 ◽

Vol 30 (2) ◽

pp. 197-211 ◽

Cited By ~ 17

Author(s):

S Chopin-Delannoy ◽

S Thenot ◽

F Delaunay ◽

E Buisine ◽

A Begue ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Nuclear Receptors ◽

Nuclear Localization ◽

Binding Domain ◽

Dna Binding Domain ◽

Protein Fusion ◽

Orphan Receptors ◽

Amino Terminal

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

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A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4507 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4507-4517 ◽

Cited By ~ 79

Author(s):

E Hadzic ◽

V Desai-Yajnik ◽

E Helmer ◽

S Guo ◽

S Wu ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Thyroid Hormone ◽

Dna Binding ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Alpha Helix ◽

Binding Domain ◽

Terminal Region ◽

Binding Studies

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

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Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.123 ◽

1993 ◽

Vol 13 (1) ◽

pp. 123-132 ◽

Cited By ~ 35

Author(s):

A D Sharrocks ◽

H Gille ◽

P E Shaw

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Serum Response Factor ◽

Alpha Helix ◽

Site Directed Mutagenesis ◽

Binding Motif ◽

Amino Acid Peptide ◽

Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

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

Eukaryotic Cell ◽

10.1128/ec.00001-09 ◽

2009 ◽

Vol 8 (4) ◽

pp. 649-664 ◽

Cited By ~ 40

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.294 ◽

1987 ◽

Vol 7 (1) ◽

pp. 294-304 ◽

Cited By ~ 30

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.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5128 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5128-5137 ◽

Cited By ~ 10

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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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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