UV photoaffinity labeling of Tn3 transposase–DNA complexes: identification of DNA binding domains

1996 ◽  
Vol 42 (1) ◽  
pp. 46-59
Author(s):  
Geoffrey S. Gottlieb ◽  
Michael A. Fennewald
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Target Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Dna Binding Domain ◽  
Transposition Frequency ◽  
Terminal Domain

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tru3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence-specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30–40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.Key words: transposon, Tn3, DNA–protein cross-linking, UV cross-linking, transposase.

scholarly journals FOXM1 Inhibitors: Emergence of a Neglected Binding Force

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Dakhili ◽  
Perez ◽  
Gopal ◽  
Ussher ◽  
Martínez
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Cancer Cell Line ◽  
Halogen Bonding ◽  
Preliminary Evidence ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Mobility Shift ◽  
Binding Interactions

The Forkhead boX M1 (FOXM1) is an essential transcription factor for normal activation of the cell cycle and cell replication. However, increasing evidence suggests that overexpression of this protein correlates with cancer development and poor patient prognosis, which makes FOXM1 a promising drug target in medicinal chemistry. Based on a computer-based molecular modeling protocol reported by our group, we hypothesized that FOXM1 inhibitors bind to the FOXM1 DNA binding domain (DBD) by (i) a pi-sulfur interaction with His287, and (ii) a halogen bonding with Arg297 within the FOXM1 DNA binding domain. To test this hypothesis, we modified the chemical structure of a known “forkhead domain inhibitor” (FDI) to synthesize and screen a series of FDI-derivatives. In this regard, we removed or replaced two essential groups in FDI-6, namely (i) the 4-fluorophenyl position and (ii) the heterocyclic sulfur atoms. We determined the inhibitory effects of test molecules on the protein expression of FOXM1 using a triple negative breast cancer cell line (MDA-MB-231), and then we measured their binding affinity to DNA by electrophoretic mobility shift assay (EMSA). Next, using a site-directed mutagenesis technique, we confirmed specific binding interactions exerted by these molecules. These results validate the role of essential binding interactions (pi-sulfur binding) predicted by computer simulations and provide preliminary evidence to postulate a mechanism of action exerted by “direct” FOXM1 inhibitors.

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.

scholarly journals Functional Domains of c-myc Promoter Binding Protein 1 Involved in Transcriptional Repression and Cell Growth Regulation

1999 ◽  
Vol 19 (4) ◽  
pp. 2880-2886 ◽  
Author(s):  
Asish K. Ghosh ◽  
Robert Steele ◽  
Ratna B. Ray
Keyword(s):  
Amino Acids ◽  
Cell Growth ◽  
Dna Binding ◽  
Transcriptional Repression ◽  
Growth Regulation ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Repressor Activity

ABSTRACT We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-mycpromoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.

A position-dependent transcription-activating domain in TFIIIA

1993 ◽  
Vol 13 (12) ◽  
pp. 7496-7506
Author(s):  
X Mao ◽  
M K Darby
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Rna Polymerase Iii ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

Molecular genetics of chromosome translocations involving EWS and related family members

1999 ◽  
Vol 1 (3) ◽  
pp. 127-138 ◽  
Author(s):  
JUNGHO KIM ◽  
JERRY PELLETIER
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Molecular Genetics ◽  
Family Members ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Chromosome Translocations ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Related Family

Kim, Jungho, and Jerry Pelletier. Molecular genetics of chromosome translocations involving EWS and related family members. Physiol. Genomics 1: 127–138, 1999.—Many types of sarcomas are characterized by specific chromosomal translocations that appear to result in the production of novel, tumor-specific chimeric transcription factors. Many of these show striking similarities: the emerging picture is that the amino-terminal domain of the fusion product is donated by the Ewing's sarcoma gene ( EWS) or a related member from the same gene family, whereas the carboxy-terminal domain often consists of a DNA-binding domain derived from one of a number of transcription factors. Given the observation that the different translocation partners of the EWS protooncogene are associated with distinct types of sarcomas, the functional consequence of fusing EWS (or a related family member) to a different DNA-binding domain can only be understood in the context of functional studies that define the specificity of action of the different fusion products. An understanding of the molecular structure and function of these translocations provides new methods for diagnosis and novel targets for therapeutics.

scholarly journals Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A ofBacillus subtilis

2000 ◽  
Vol 182 (24) ◽  
pp. 6975-6982 ◽  
Author(s):  
Janet K. Hatt ◽  
Philip Youngman
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Response Regulator ◽  
Gene Activation ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Specific Gene ◽  
Dna Binding Domain ◽  
Conserved Residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

scholarly journals Two Domains Unique to Osteoblast-Specific Transcription Factor Osf2/Cbfa1 Contribute to Its Transactivation Function and Its Inability To Heterodimerize with Cbfβ

1998 ◽  
Vol 18 (7) ◽  
pp. 4197-4208 ◽  
Author(s):  
Kannan Thirunavukkarasu ◽  
Muktar Mahajan ◽  
Keith W. McLarren ◽  
Stefano Stifani ◽  
Gerard Karsenty
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Critical Role ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Amino Terminal ◽  
Related Proteins ◽  
Repression Domain

ABSTRACT Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfβ, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

scholarly journals Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

2007 ◽  
Vol 403 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Nathalie Gillard ◽  
Stephane Goffinont ◽  
Corinne Buré ◽  
Marie Davidkova ◽  
Jean-Claude Maurizot ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Specific Binding ◽  
Classical Model ◽  
Binding Domain ◽  
Oh Radicals ◽  
Water Radiolysis ◽  
Dna Binding Domain ◽  
Fluorescence Measurements ◽  
Radiation Induced

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

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