scholarly journals DNA-binding specificity of the cut repeats from the human cut-like protein.

1995 ◽  
Vol 15 (1) ◽  
pp. 129-140 ◽  
Author(s):  
R Harada ◽  
G Bérubé ◽  
O J Tamplin ◽  
C Denis-Larose ◽  
A Nepveu
Keyword(s):  
Dna Binding ◽  
Electrophoretic Mobility Shift Assay ◽  
Fusion Proteins ◽  
Glutathione S Transferase ◽  
Mobility Shift ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
A Site

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

scholarly journals DNA-binding specificity of GATA family transcription factors

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

1993 ◽  
Vol 13 (2) ◽  
pp. 852-860
Author(s):  
M B Toledano ◽  
D Ghosh ◽  
F Trinh ◽  
W J Leonard
Keyword(s):  
Dna Binding ◽  
Point Mutations ◽  
Terminal Region ◽  
Sequence Motif ◽  
Structural Determinants ◽  
Nf Kappa B ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Specificity

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

scholarly journals Specific Binding of Integrase to the Origin of Transfer (oriT) of the Conjugative Transposon Tn916

2001 ◽  
Vol 183 (9) ◽  
pp. 2947-2951 ◽  
Author(s):  
Douglas Hinerfeld ◽  
Gordon Churchward
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Conjugal Transfer ◽  
Dna Binding Domain ◽  
Conjugative Transposon ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
A Site ◽  
Integrase Protein

ABSTRACT Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding tooriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

scholarly journals Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1123-1133 ◽  
Author(s):  
Jay L. Mellies ◽  
Gregory Benison ◽  
William McNitt ◽  
David Mavor ◽  
Chris Boniface ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Effector Proteins ◽  
Watery Diarrhoea ◽  
Mobility Shift ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Secretory Diarrhoea ◽  
Mobility Shift Assay ◽  
Enterocyte Effacement

Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler–DNA structures that are predicted to be involved in stimulating gene expression.

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