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PDBsum entry 1d66
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protein dna_rna metals Protein-protein interface(s) links
Transcription/DNA PDB id
1d66

 

 

 

 

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Contents
Protein chains
57 a.a. *
DNA/RNA
Metals
_CD ×4
Waters ×51
* Residue conservation analysis
PDB id:
1d66
Name: Transcription/DNA
Title: DNA recognition by gal4: structure of a protein/DNA complex
Structure: DNA (5'- d( Cp Cp Gp Gp Ap Gp Gp Ap Cp Ap Gp Tp Cp Cp Tp Cp C p Gp G)-3'). Chain: d. Engineered: yes. DNA (5'- d( Cp Cp Gp Gp Ap Gp Gp Ap Cp Tp Gp Tp Cp Cp Tp Cp C p Gp G)-3'). Chain: e. Engineered: yes. Protein (gal4).
Source: Synthetic: yes. Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Dimer (from PQS)
Resolution:
2.70Å     R-factor:   0.230    
Authors: R.Marmorstein,M.Carey,M.Ptashne,S.C.Harrison
Key ref: R.Marmorstein et al. (1992). DNA recognition by GAL4: structure of a protein-DNA complex. Nature, 356, 408-414. PubMed id: 1557122
Date:
06-Mar-92     Release date:   06-Mar-92    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P04386  (GAL4_YEAST) -  Regulatory protein GAL4 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Seq:
Struc: 		 
Seq:
Struc: 		881 a.a.
57 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

DNA/RNA chains
  C-C-G-G-A-G-G-A-C-A-G-T-C-C-T-C-C-G-G 19 bases
  C-C-G-G-A-G-G-A-C-T-G-T-C-C-T-C-C-G-G 19 bases

 Enzyme reactions 
   Enzyme class: E.C.?
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

 

 
Nature 356:408-414 (1992)
PubMed id: 1557122  
 
 
DNA recognition by GAL4: structure of a protein-DNA complex.
R.Marmorstein, M.Carey, M.Ptashne, S.C.Harrison.
 
  ABSTRACT  
 
A specific DNA complex of the 65-residue, N-terminal fragment of the yeast transcriptional activator, GAL4, has been analysed at 2.7 A resolution by X-ray crystallography. The protein binds as a dimer to a symmetrical 17-base-pair sequence. A small, Zn(2+)-containing domain recognizes a conserved CCG triplet at each end of the site through direct contacts with the major groove. A short coiled-coil dimerization element imposes 2-fold symmetry. A segment of extended polypeptide chain links the metal-binding module to the dimerization element and specifies the length of the site. The relatively open structure of the complex would allow another protein to bind coordinately with GAL4.
 

Literature references that cite this PDB file's key reference

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DNA-carrier proteins for targeted gene delivery.
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PDB code: 1lpv
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Cross-pathway regulation in Saccharomyces cerevisiae: activation of the proline utilization pathway by Ga14p in vivo.
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An overview of the structures of protein-DNA complexes.
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Regulation of pleiotropic drug resistance in yeast.
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Deregulation of gluconeogenic structural genes by variants of the transcriptional activator Cat8p of the yeast Saccharomyces cerevisiae.
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Binding of Gal4p and bicoid to nucleosomal sites in yeast in the absence of replication.
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The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex.
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Transcriptional induction by aromatic amino acids in Saccharomyces cerevisiae.
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10092669 I.Nikolaev, F.Lenouvel, and B.Felenbok (1999).
Unique DNA binding specificity of the binuclear zinc AlcR activator of the ethanol utilization pathway in Aspergillus nidulans.
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10216264 K.C.Ehrlich, B.G.Montalbano, and J.W.Cary (1999).
Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus.
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10026283 K.Nadassy, S.J.Wodak, and J.Janin (1999).
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10358080 P.C.Liu, and D.J.Thiele (1999).
Modulation of human heat shock factor trimerization by the linker domain.
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Function and regulation of yeast hexose transporters.
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Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation.
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10428861 T.Hon, A.Hach, D.Tamalis, Y.Zhu, and L.Zhang (1999).
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Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans.
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The use of variable density self-assembled monolayers to probe the structure of a target molecule.
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Structure-based design of a dimeric zinc finger protein.
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9852083 J.M.Cánaves, and M.Montal (1998).
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The regulator of nitrate assimilation in ascomycetes is a dimer which binds a nonrepeated, asymmetrical sequence.
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An analysis of the relationship between hydration and protein-DNA interactions.
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Kluyveromyces lactis SEF1 and its Saccharomyces cerevisiae homologue bypass the unknown essential function, but not the mitochondrial RNase P function, of the S. cerevisiae RPM2 gene.
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Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1.
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9466262 N.N.van Peij, J.Visser, and L.H.de Graaff (1998).
Isolation and analysis of xlnR, encoding a transcriptional activator co-ordinating xylanolytic expression in Aspergillus niger.
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Structural dissection of the DNA-binding domain of the yeast transcriptional activator GAL4 reveals an alpha-helical region responsible for dimerization.
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FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences.
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Joint molecular modeling and spectroscopic studies of DNA complexes of a bis(arginyl) conjugate of a tricationic porphyrin designed to target the major groove.
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Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain.
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Structures of SAP-1 bound to DNA targets from the E74 and c-fos promoters: insights into DNA sequence discrimination by Ets proteins.
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PDB codes: 1bc7 1bc8
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NMR analysis of CYP1(HAP1) DNA binding domain-CYC1 upstream activation sequence interactions: recognition of a CGG trinucleotide and of an additional thymine 5 bp downstream by the zinc cluster and the N-terminal extremity of the protein.
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9278448 C.Panozzo, V.Capuano, S.Fillinger, and B.Felenbok (1997).
The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans.
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Probing the architecture of a simple kinetochore using DNA-protein crosslinking.
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High-affinity binding of the cell cycle-regulated transcription factors E2F1 and E2F4 to benzo[a]pyrene diol epoxide-DNA adducts.
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Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae.
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Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p.
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9659911 G.Mizuguchi, T.Tsukiyama, J.Wisniewski, and C.Wu (1997).
Role of nucleosome remodeling factor NURF in transcriptional activation of chromatin.
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Eukaryotic transcription factor-DNA complexes.
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Structure and mobility of the PUT3 dimer.
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PDB code: 1ajy
9303004 K.Swaminathan, P.Flynn, R.J.Reece, and R.Marmorstein (1997).
Crystal structure of a PUT3-DNA complex reveals a novel mechanism for DNA recognition by a protein containing a Zn2Cys6 binuclear cluster.
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PDB code: 1zme
9236000 M.M.Javadpour, and M.D.Barkley (1997).
Self-assembly of designed antimicrobial peptides in solution and micelles.
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9782776 R.E.Dickerson, and T.K.Chiu (1997).
Helix bending as a factor in protein/DNA recognition.
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9118994 R.Näit-Kaoudjt, R.Williams, B.Guiard, and M.Gervais (1997).
Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities.
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SWI/SNF stimulates the formation of disparate activator-nucleosome complexes but is partially redundant with cooperative binding.
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9228952 S.F.Bellon, K.K.Rodgers, D.G.Schatz, J.E.Coleman, and T.A.Steitz (1997).
Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster.
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PDB code: 1rmd
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Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.
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9241414 V.Ramakrishnan (1997).
Histone structure and the organization of the nucleosome.
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Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4.
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The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals.
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9261070 Y.Tao, S.V.Strelkov, V.V.Mesyanzhinov, and M.G.Rossmann (1997).
Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.
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PDB code: 1aa0
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Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae.
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8757934 J.C.Drapier, and C.Bouton (1996).
Modulation by nitric oxide of metalloprotein regulatory activities.
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Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions.
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A novel DNA binding motif for yeast zinc cluster proteins: the Leu3p and Pdr3p transcriptional activators recognize everted repeats.
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Factors influencing accuracy of computer-built models: a study based on leucine zipper GCN4 structure.
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Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro.
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The Saccharomyces cerevisiae kinetochore contains a cyclin-CDK complexing homologue, as identified by in vitro reconstitution.
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Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators.
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Boehringer Mannheim award lecture 1995. La conference Boehringer Mannheim 1995. De novo design of alpha-helical proteins: basic research to medical applications.
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DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA.
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Cha4p of Saccharomyces cerevisiae activates transcription via serine/threonine response elements.
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Two different repressors collaborate to restrict expression of the yeast glucose transporter genes HXT2 and HXT4 to low levels of glucose.
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8668125 W.Jiang, and Y.Koltin (1996).
Two-hybrid interaction of a human UBC9 homolog with centromere proteins of Saccharomyces cerevisiae.
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Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein.
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Contact with a component of the polymerase II holoenzyme suffices for gene activation.
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Gene regulatory proteins and their interaction with DNA.
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Structures of protein complexes by multidimensional heteronuclear magnetic resonance spectroscopy.
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7876302 A.V.Strunnikov, J.Kingsbury, and D.Koshland (1995).
CEP3 encodes a centromere protein of Saccharomyces cerevisiae.
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Two mutually exclusive regulatory systems inhibit UASGATA, a cluster of 5'-GAT(A/T)A-3' upstream from the UGA4 gene of Saccharomyces cerevisiae.
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Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.
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Cis- and trans-acting elements determining induction of the genes of the gamma-aminobutyrate (GABA) utilization pathway in Saccharomyces cerevisiae.
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8529657 E.M.Friedl, and P.Matthias (1995).
Transcriptional activation and repression, two properties of the lymphoid-specific transcription factor Oct-2a.
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Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.
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A 29.425 kb segment on the left arm of yeast chromosome XV contains more than twice as many unknown as known open reading frames.
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7716167 J.Janin (1995).
Elusive affinities.
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Solution structure of the Kluyveromyces lactis LAC9 Cd2 Cys6 DNA-binding domain.
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PDB code: 1cld
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The DNA binding domains of the yeast Gal4 and human c-Jun transcription factors interact through the zinc-finger and bZIP motifs.
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Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.
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PDB code: 2abk
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Sequence-specific DNA binding by covalently constrained peptide dimers of the basic leucine zipper protein GCN4.
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Binding geometry of alpha-helices that recognize DNA.
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Stereochemical basis of DNA bending by transcription factors.
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The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.
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8618837 P.K.Sorger, K.F.Doheny, P.Hieter, K.M.Kopski, T.C.Huffaker, and A.A.Hyman (1995).
Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.
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UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner.
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A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg).
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The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae.
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8750235 V.V.Svetlov, and T.G.Cooper (1995).
Review: compilation and characteristics of dedicated transcription factors in Saccharomyces cerevisiae.
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7596294 Y.H.Fu, B.Feng, S.Evans, and G.A.Marzluf (1995).
Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora.
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A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.
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Phenotypic mixing between different hepadnavirus nucleocapsid proteins reveals C protein dimerization to be cis preferential.
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Approaching the function of new genes by detection of their potential upstream activation sequences in Saccharomyces cerevisiae: application to chromosome III.
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Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis.
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Heterodimerization of the yeast MATa1 and MAT alpha 2 proteins is mediated by two leucine zipper-like coiled-coil motifs.
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9383374 D.A.Erlanson, and G.L.Verdine (1994).
Falling out of the fold: tumorigenic mutations and p53.
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Transcriptional control of the yeast PDR5 gene by the PDR3 gene product.
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Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.
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Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP.
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PDB code: 1ctl
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Young Investigator Award Lecture. Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR.
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The DNA-binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster.
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A zinc finger protein, essential for chromosome segregation, constitutes a putative DNA binding subunit of the Saccharomyces cerevisiae kinetochore complex, Cbf3.
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7854115 J.Pfau, D.N.Arvidson, and P.Youderian (1994).
Mutants of Escherichia coli Trp repressor with changes of conserved, helix-turn-helix residue threonine 81 have altered DNA-binding specificities.
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Zinc mining for protein domains.
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Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities.
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Differential repression of transcription factor binding by histone H1 is regulated by the core histone amino termini.
  EMBO J, 13, 6031-6040.  
8041727 L.Nekludova, and C.O.Pabo (1994).
Distinctive DNA conformation with enlarged major groove is found in Zn-finger-DNA and other protein-DNA complexes.
  Proc Natl Acad Sci U S A, 91, 6948-6952.  
8052649 M.B.Cachon-Gonzalez, S.Fenner, J.M.Coffin, C.Moran, S.Best, and J.P.Stoye (1994).
Structure and expression of the hairless gene of mice.
  Proc Natl Acad Sci U S A, 91, 7717-7721.  
8087558 M.Suzuki (1994).
A framework for the DNA-protein recognition code of the probe helix in transcription factors: the chemical and stereochemical rules.
  Structure, 2, 317-326.  
  8065339 M.Tanaka, and W.Herr (1994).
Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.
  Mol Cell Biol, 14, 6056-6067.  
  8289837 M.Vettese-Dadey, P.Walter, H.Chen, L.J.Juan, and J.L.Workman (1994).
Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores.
  Mol Cell Biol, 14, 970-981.  
  8035807 S.Chaudhary, C.Brou, M.E.Valentin, N.Burton, L.Tora, P.Chambon, and I.Davidson (1994).
A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1.
  Mol Cell Biol, 14, 5290-5299.  
  8139572 Y.Ohashi, J.M.Brickman, E.Furman, B.Middleton, and M.Carey (1994).
Modulating the potency of an activator in a yeast in vitro transcription system.
  Mol Cell Biol, 14, 2731-2739.  
8396762 C.Brou, A.Kuhn, A.Staub, S.Chaudhary, I.Grummt, I.Davidson, and L.Tora (1993).
Sequence-specific transactivators counteract topoisomerase II-mediated inhibition of in vitro transcription by RNA polymerases I and II.
  Nucleic Acids Res, 21, 4011-4018.  
8327484 G.Fang, and T.R.Cech (1993).
Oxytricha telomere-binding protein: DNA-dependent dimerization of the alpha and beta subunits.
  Proc Natl Acad Sci U S A, 90, 6056-6060.  
  8467796 G.Stone, and I.Sadowski (1993).
GAL4 is regulated by a glucose-responsive functional domain.
  EMBO J, 12, 1375-1385.  
7763979 J.A.Tainer, and R.P.Cunningham (1993).
Molecular recognition in DNA-binding proteins and enzymes.
  Curr Opin Biotechnol, 4, 474-483.  
7763973 J.G.Adamson, N.E.Zhou, and R.S.Hodges (1993).
Structure, function and application of the coiled-coil protein folding motif.
  Curr Opin Biotechnol, 4, 428-437.  
  8428590 J.H.Carra, and R.F.Schleif (1993).
Variation of half-site organization and DNA looping by AraC protein.
  EMBO J, 12, 35-44.  
  8423776 J.Sidorova, and L.Breeden (1993).
Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.
  Mol Cell Biol, 13, 1069-1077.  
8506279 J.W.Michelsen, K.L.Schmeichel, M.C.Beckerle, and D.R.Winge (1993).
The LIM motif defines a specific zinc-binding protein domain.
  Proc Natl Acad Sci U S A, 90, 4404-4408.  
  8355711 J.Y.Sze, E.Remboutsika, and G.B.Kohlhaw (1993).
Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.
  Mol Cell Biol, 13, 5702-5709.  
8440748 L.P.Freedman, and B.F.Luisi (1993).
On the mechanism of DNA binding by nuclear hormone receptors: a structural and functional perspective.
  J Cell Biochem, 51, 140-150.  
8464899 L.Zhang, O.Bermingham-McDonogh, B.Turcotte, and L.Guarente (1993).
Antibody-promoted dimerization bypasses the regulation of DNA binding by the heme domain of the yeast transcriptional activator HAP1.
  Proc Natl Acad Sci U S A, 90, 2851-2855.  
8119589 M.A.Davis, J.M.Kelly, and M.J.Hynes (1993).
Fungal catabolic gene regulation: molecular genetic analysis of the amdS gene of Aspergillus nidulans.
  Genetica, 90, 133-145.  
  8491199 M.Molinete, W.Vermeulen, A.Bürkle, J.Ménissier-de Murcia, J.H.Küpper, J.H.Hoeijmakers, and G.de Murcia (1993).
Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.
  EMBO J, 12, 2109-2117.  
8317827 P.S.Freemont (1993).
The RING finger. A novel protein sequence motif related to the zinc finger.
  Ann N Y Acad Sci, 684, 174-192.  
7681583 R.Lovering, I.M.Hanson, K.L.Borden, S.Martin, N.J.O'Reilly, G.I.Evan, D.Rahman, D.J.Pappin, J.Trowsdale, and P.S.Freemont (1993).
Identification and preliminary characterization of a protein motif related to the zinc finger.
  Proc Natl Acad Sci U S A, 90, 2112-2116.  
  8458333 S.Fytlovich, M.Gervais, C.Agrimonti, and B.Guiard (1993).
Evidence for an interaction between the CYP1(HAP1) activator and a cellular factor during heme-dependent transcriptional regulation in the yeast Saccharomyces cerevisiae.
  EMBO J, 12, 1209-1218.  
  8395001 S.Walker, R.Greaves, and P.O'Hare (1993).
Transcriptional activation by the acidic domain of Vmw65 requires the integrity of the domain and involves additional determinants distinct from those necessary for TFIIB binding.
  Mol Cell Biol, 13, 5233-5244.  
  8445719 W.Sun, M.O'Connell, and N.A.Speck (1993).
Characterization of a protein that binds multiple sequences in mammalian type C retrovirus enhancers.
  J Virol, 67, 1976-1986.  
  1620111 E.A.Golemis, and R.Brent (1992).
Fused protein domains inhibit DNA binding by LexA.
  Mol Cell Biol, 12, 3006-3014.  
1368439 J.A.Tainer, V.A.Roberts, and E.D.Getzoff (1992).
Protein metal-binding sites.
  Curr Opin Biotechnol, 3, 378-387.  
  1280325 J.G.Kim, and L.D.Hudson (1992).
Novel member of the zinc finger superfamily: A C2-HC finger that recognizes a glia-specific gene.
  Mol Cell Biol, 12, 5632-5639.  
  1323708 J.M.Hardwick, L.Tse, N.Applegren, J.Nicholas, and M.A.Veliuona (1992).
The Epstein-Barr virus R transactivator (Rta) contains a complex, potent activation domain with properties different from those of VP16.
  J Virol, 66, 5500-5508.  
15336020 J.W.Schwabe (1992).
DNA between the sheets.
  Curr Biol, 2, 661-663.  
  1464322 K.H.Emami, and M.Carey (1992).
A synergistic increase in potency of a multimerized VP16 transcriptional activation domain.
  EMBO J, 11, 5005-5012.  
1479891 L.M.Parsons, M.A.Davis, and M.J.Hynes (1992).
Identification of functional regions of the positively acting regulatory gene amdR from Aspergillus nidulans.
  Mol Microbiol, 6, 2999-3007.  
  1448098 M.R.Parthun, D.A.Mangus, and J.A.Jaehning (1992).
The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.
  Mol Cell Biol, 12, 5683-5689.  
  1304897 P.Bork, C.Ouzounis, C.Sander, M.Scharf, R.Schneider, and E.Sonnhammer (1992).
Comprehensive sequence analysis of the 182 predicted open reading frames of yeast chromosome III.
  Protein Sci, 1, 1677-1690.  
1429878 T.Boulikas (1992).
Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.
  J Cell Biochem, 50, 111-123.  
  1303761 T.Mau, J.D.Baleja, and G.Wagner (1992).
Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.
  Protein Sci, 1, 1403-1412.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.

 

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