Nejvíce citovaný článek - PubMed ID 20334529
The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.
- MeSH
- Bacillus subtilis * genetika metabolismus MeSH
- bakteriální proteiny * chemie metabolismus genetika MeSH
- DNA bakterií metabolismus chemie genetika MeSH
- DNA vazebné proteiny chemie metabolismus genetika MeSH
- DNA chemie metabolismus MeSH
- elektronová kryomikroskopie * MeSH
- fruktosadifosfáty MeSH
- krystalografie rentgenová MeSH
- molekulární modely * MeSH
- multimerizace proteinu MeSH
- operon genetika MeSH
- regulace genové exprese u bakterií MeSH
- represorové proteiny * chemie metabolismus genetika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny * MeSH
- DNA bakterií MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- fructose-1,6-diphosphate MeSH Prohlížeč
- fruktosadifosfáty MeSH
- represorové proteiny * MeSH
Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
- Klíčová slova
- DNA polymerases, bioconjugations, cross-linking reactions, nucleotides, proteins,
- MeSH
- arginin chemie MeSH
- DNA chemická syntéza chemie MeSH
- histony chemie MeSH
- ketony chemická syntéza chemie MeSH
- nádorový supresorový protein p53 chemie MeSH
- peptidy chemie MeSH
- proteiny chemie MeSH
- reagencia zkříženě vázaná chemická syntéza chemie MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- thiminnukleotidy chemická syntéza chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- arginin MeSH
- DNA MeSH
- histony MeSH
- ketony MeSH
- nádorový supresorový protein p53 MeSH
- peptidy MeSH
- proteiny MeSH
- reagencia zkříženě vázaná MeSH
- sérový albumin hovězí MeSH
- thiminnukleotidy MeSH
Interaction with the DNA minor groove is a significant contributor to specific sequence recognition in selected families of DNA-binding proteins. Based on a statistical analysis of 3D structures of protein-DNA complexes, we propose that distortion of the DNA minor groove resulting from interactions with hydrophobic amino acid residues is a universal element of protein-DNA recognition. We provide evidence to support this by associating each DNA minor groove-binding amino acid residue with the local dimensions of the DNA double helix using a novel algorithm. The widened DNA minor grooves are associated with high GC content. However, some AT-rich sequences contacted by hydrophobic amino acids (e.g., phenylalanine) display extreme values of minor groove width as well. For a number of hydrophobic amino acids, distinct secondary structure preferences could be identified for residues interacting with the widened DNA minor groove. These results hold even after discarding the most populous families of minor groove-binding proteins.
- Klíčová slova
- DNA shape, hydrophobic, indirect readout, minor groove, protein–DNA interaction, specific recognition,
- MeSH
- algoritmy MeSH
- aminokyselinové motivy MeSH
- aminokyseliny chemie MeSH
- Arabidopsis metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA chemie MeSH
- fenylalanin chemie MeSH
- hydrofobní a hydrofilní interakce * MeSH
- konformace nukleové kyseliny MeSH
- kyselina glutamová chemie MeSH
- lidé MeSH
- proteiny chemie MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- sekundární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- fenylalanin MeSH
- kyselina glutamová MeSH
- proteiny MeSH
Squaramate-linked 2'-deoxycytidine 5'-O-triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate-linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys-containing peptides. Squaramate-linked DNA formed covalent cross-links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.
- Klíčová slova
- DNA, DNA polymerase, bioconjugation, cross-linking reactions, proteins,
- MeSH
- DNA metabolismus MeSH
- lidé MeSH
- lysin metabolismus MeSH
- nukleotidy metabolismus MeSH
- peptidy chemie MeSH
- proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- lysin MeSH
- nukleotidy MeSH
- peptidy MeSH
- proteiny MeSH
We analyzed the structural behavior of DNA complexed with regulatory proteins and the nucleosome core particle (NCP). The three-dimensional structures of almost 25 thousand dinucleotide steps from more than 500 sequentially non-redundant crystal structures were classified by using DNA structural alphabet CANA (Conformational Alphabet of Nucleic Acids) and associations between ten CANA letters and sixteen dinucleotide sequences were investigated. The associations showed features discriminating between specific and non-specific binding of DNA to proteins. Important is the specific role of two DNA structural forms, A-DNA, and BII-DNA, represented by the CANA letters AAA and BB2: AAA structures are avoided in non-specific NCP complexes, where the wrapping of the DNA duplex is explained by the periodic occurrence of BB2 every 10.3 steps. In both regulatory and NCP complexes, the extent of bending of the DNA local helical axis does not influence proportional representation of the CANA alphabet letters, namely the relative incidences of AAA and BB2 remain constant in bent and straight duplexes.
- Klíčová slova
- DNA, DNA-protein recognition, histone, molecular structure, nucleosome core particle, regulatory proteins, transcription factors,
- Publikační typ
- časopisecké články MeSH
Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue-amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein-DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties.
- MeSH
- adenin chemie metabolismus MeSH
- aminokyselinové motivy * MeSH
- aminokyseliny chemie metabolismus MeSH
- cytosin chemie metabolismus MeSH
- databáze proteinů MeSH
- DNA vazebné proteiny chemie genetika metabolismus MeSH
- DNA chemie genetika metabolismus MeSH
- guanin chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- statistika jako téma metody MeSH
- terciární struktura proteinů MeSH
- termodynamika MeSH
- thymin chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- adenin MeSH
- aminokyseliny MeSH
- cytosin MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- guanin MeSH
- thymin MeSH
Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over- or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure.
- MeSH
- biomechanika MeSH
- DNA chemie MeSH
- dvouvláknová RNA chemie MeSH
- konformace nukleové kyseliny MeSH
- párování bází MeSH
- simulace molekulární dynamiky MeSH
- točivý moment MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA MeSH
- dvouvláknová RNA MeSH
The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE.
- MeSH
- DNA chemie MeSH
- element responzivní pro sérum genetika MeSH
- fosfáty chemie MeSH
- genetická transkripce MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nukleotidové motivy MeSH
- oligonukleotidy chemie MeSH
- Ramanova spektroskopie MeSH
- rekombinantní proteiny chemie genetika MeSH
- serum response factor chemie genetika MeSH
- simulace molekulární dynamiky MeSH
- statická elektřina MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- fosfáty MeSH
- oligonukleotidy MeSH
- rekombinantní proteiny MeSH
- serum response factor MeSH
- SRF protein, human MeSH Prohlížeč
- MeSH
- biosenzitivní techniky přístrojové vybavení metody MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- glykomika přístrojové vybavení metody MeSH
- glykoproteiny analýza metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- proteiny analýza metabolismus MeSH
- sacharidové sekvence MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- glykoproteiny MeSH
- proteiny MeSH
To investigate the principles driving recognition between proteins and DNA, we analyzed more than thousand crystal structures of protein/DNA complexes. We classified protein and DNA conformations by structural alphabets, protein blocks [de Brevern, Etchebest and Hazout (2000) (Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks. Prots. Struct. Funct. Genet., 41:271-287)] and dinucleotide conformers [Svozil, Kalina, Omelka and Schneider (2008) (DNA conformations and their sequence preferences. Nucleic Acids Res., 36:3690-3706)], respectively. Assembling the mutually interacting protein blocks and dinucleotide conformers into 'interaction matrices' revealed their correlations and conformer preferences at the interface relative to their occurrence outside the interface. The analyzed data demonstrated important differences between complexes of various types of proteins such as transcription factors and nucleases, distinct interaction patterns for the DNA minor groove relative to the major groove and phosphate and importance of water-mediated contacts. Water molecules mediate proportionally the largest number of contacts in the minor groove and form the largest proportion of contacts in complexes of transcription factors. The generally known induction of A-DNA forms by complexation was more accurately attributed to A-like and intermediate A/B conformers rare in naked DNA molecules.
- MeSH
- DNA vazebné proteiny chemie MeSH
- DNA chemie MeSH
- fosfáty MeSH
- interpretace statistických dat MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- vazba proteinů MeSH
- voda chemie MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA MeSH
- fosfáty MeSH
- voda MeSH