Protein-nucleic Acid Complexes Research Articles

Molecular dynamics simulation of nucleic acids.

We review molecular dynamics simulations of nucleic acids, including those completed from 1995 to 2000, with a focus on the applications and results rather than the methods. After the introduction, which discusses recent advances in the simulation of nucleic acids in solution, we describe force fields for nucleic acids and then provide a detailed summary of the published literature. We emphasize simulations of small nucleic acids ( approximately 6 to 24 mer) in explicit solvent with counterions, using reliable force fields and modern simulation protocols that properly represent the long-range electrostatic interactions. We also provide some limited discussion of simulation in the absence of explicit solvent. Absent from this discussion are results from simulations of protein-nucleic acid complexes and modified DNA analogs. Highlights from the molecular dynamics simulation are the spontaneous observation of A B transitions in duplex DNA in response to the environment, specific ion binding and hydration, and reliable representation of protein-nucleic acid interactions. We close by examining major issues and the future promise for these methods.

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

Background: This study addresses the general problem of dividing a density map of a nucleic-acid–protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches ∼3 Å it is generally possible to interpret its shape (i.e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. Results: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 Å resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. Conclusions: The new method, when applied to an 11.5 Å cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.

Caspase-dependent cleavage of nucleic acids.

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production. While apoptosis is specifically characterized by posttranslational modification of proteins, recent findings also show that nucleic acids are modified. This review summarizes the specific cleavages of some of these key nucleic acids, i.e. chromosomal DNA, ribosomal RNA and small structural RNAs (U1 snRNA, Y RNA), in apoptotic cells.

NMR spectroscopy: a multifaceted approach to macromolecular structure.

1. Introduction 292. Landmarks in NMR of macromolecules 322.1 Protein structures and methods development 322.1.1 Sequential assignment method 322.1.2 Triple-resonance experiments 342.1.3 Structures of large proteins 362.2 Protein–nucleic acid complexes 372.3 RNA structures 382.4 Membrane-bound systems 393. NMR spectroscopy today 403.1 State-of-the-art structure determination 413.2 New methods 443.2.1 Residual dipolar couplings 443.2.2 Direct detection of hydrogen bonds 443.2.3 Spin labeling 453.2.4 Segmental labeling 463.3 Protein complexes 473.4 Mobility studies 503.5 Determination of time-dependent structures 523.6 Drug discovery 534. The future of NMR 544.1 The ease of structure determination 544.2 The ease of making recombinant protein 554.3 Post-translationally modified proteins 554.4 Approaches to large and/or membrane-bound proteins 564.5 NMR in structural genomics 564.6 Synergy of NMR and crystallography in protein structure determination 565. Conclusion 576. Acknowledgements 577. References 57Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dötsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

Use of Biotin-Labeled Nucleic Acids for Protein Purification and Agarose-Based Chemiluminescent Electromobility Shift Assays

We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin–agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein–RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein–nucleic acid complexes. After separation of free from complexed RNA in agarose, the RNA is electroblotted to positively charged nylon. The biotin-labeled RNA is readily bound by a streptavidin–alkaline phosphatase conjugate, allowing for very sensitive chemiluminescent detection (∼0.1–1.0 fmol limit). Using our system, we were able to purify both known iron-responsive proteins (IRPs) from rat liver and assess their binding affinity to RNA containing the iron-responsive element (IRE) using the same batch of biotinylated RNA. We show data indicating that agarose is especially useful for cases when large complexes are formed, although smaller complexes are even better resolved.

Sulfur-containing Nucleic Acids: Synthesis, Chemical Reactions in Supramolecular Biopolymer Complexes, Biological Applications

The chemical behavior of sulfur-containing oligonucleotides and their reactivity in self-assembled nucleic acids (NA) and specific NA–protein complexes is considered. Reviewed are postsynthetic approaches that allow introducing sulfur-containing linkages at preselected positions of the sugar-phosphate backbone of DNA and between neighboring nucleobases, to incorporate disulfide bridges between complementary strands of double- and triple-stranded DNAs, in large catalytic RNA, etc. Special reference is given to the site-specific chemical modifications as a tool for elucidating the structure, folding, and function of biomolecules. Structure-directed chemical reactions are shown to be helpful in detecting point mutations in DNA, targeting the modifications on specific positions of NA, probing the molecular recognition in protein–DNA interfaces, studying the conformational dynamics of nucleic acids, and discriminating between different folding models.

1L1715 蛋白質-核酸相互作用の熱力学データベース

Protein-nucleic acid interaction plays an essential role in the regulation of gene expression. Structural and binding information provides insight into the mechanism of protein-nucleic acid recognition. Although there are structural databases for protein-nucleic acid complexes, there exists no database for protein-nucleic acid binding. Thus, we have decided to develop the Thermodynamic Database for Protein-Nucleic Acid Interactions. It contains several important thermodynamic parameters, experimental conditions, structural information about proteins, nucleic acids and of the complex, and literature information. This database will be helpful for understanding the mechanism of proteinnucleic acid recognition. A WWW interface will help users to search the data based on various conditions with different sorting options for outputs.

Recognition of an Essential Adenine at a Protein−RNA Interface: Comparison of the Contributions of Hydrogen Bonds and a Stacking Interaction

Recognition of an Essential Adenine at a Protein−RNA Interface: Comparison of the Contributions of Hydrogen Bonds and a Stacking Interaction

High Precision Solution Structure of the C-terminal KH Domain of Heterogeneous Nuclear Ribonucleoprotein K, a c-myc Transcription Factor

Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26→Arg mutant of the C-terminal K-homology (KH) domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded β-sheet stacked against three α-helices, two of which are nearly parallel to the strands of the β-sheet. The Gly26→Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the β-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.

HCCCH Experiment for Through-Bond Correlation of Thymine Resonances in13C-Labeled DNA Oligonucleotides

Application of heteronuclear magnetic resonance pulse methods to13C,15N-labeled nucleic acids is important for the accurate structure determination of larger RNA and DNA oligonucleotides and protein–nucleic acid complexes. These methods have been applied primarily to RNA, due to the availability of labeled samples. The two major differences between DNA and RNA are at the C2′ of the ribose and deoxyribose and the additional methyl group on thymine versus uracil. We have enzymatically synthesized a13C,15N-labeled 32 base DNA oligonucleotide that folds to form an intramolecular triplex. We present two- and three-dimensional versions of a new HCCCH–TOCSY experiment that provides intraresidue correlation between the thymine H6 and methyl resonances via the intervening carbons (H6–C6–C5–Cme–Hme).

NMR studies of protein-nucleic acid complexes: structures, solvation, dynamics and coupled protein folding.

Many basic events which concern management and manipulation of genes within a cell – for instance transcription, replication and recombination – rely on specific interactions between proteins and nucleic acids. Such interactions are also essential for many house-keeping functions, like packing and unpacking of DNA in chromatin and assembly of ribosomes. Moreover, the details of protein–nucleic acid interplay is essential for understanding the action of viruses. The list of functional mechanisms in biology that rely on protein–DNA and protein–RNA interactions can be made much longer, but these examples represent some of the topics which motivated structural biologists to study complexes between proteins and nucleic acids as a first step beyond structure determinations of individual biomolecules.

Structural features of protein-nucleic acid recognition sites.

We analyzed the atomic models of 75 X-ray structures of protein-nucleic acid complexes with the aim of uncovering common properties. The interface area measured the extent of contact between the protein and nucleic acid. It was found to vary between 1120 and 5800 A2. Despite this wide variation, the interfaces in complexes of transcription factors with double-stranded DNA could be broken up into recognition modules where 12 +/- 3 nucleotides on the DNA side contact 24 +/- 6 amino acids on the protein side, with interface areas in the range 1600 +/- 400 A2. For enzymes acting on DNA, the recognition module is on average 600 A2 larger, due to the requirement of making an active site. As judged by its chemical and amino acid composition, the average protein surface in contact with the DNA is more polar than the solvent accessible surface or the typical protein-protein interface. The protein side is rich in positively charged groups from lysine and arginine side chains; on the DNA side the negative charges from phosphate groups dominate. Hydrogen bonding patterns were also analyzed, and we found one intermolecular hydrogen bond per 125 A2 of interface area in high-resolution structures. An equivalent number of polar interactions involved water molecules, which are generally abundant at protein-DNA interfaces. Calculations of Voronoi atomic volumes, performed in the presence and absence of water molecules, showed that protein atoms buried at the interface with DNA are on average as closely packed as in the protein interior. Water molecules contribute to the close packing, thereby mediating shape complementarity. Finally, conformational changes accompanying association were analyzed in 24 of the complexes for which the structure of the free protein was also available. On the DNA side the extent of deformation showed some correlation with the size of the interface area. On the protein side the type and size of the structural changes spanned a wide spectrum. Disorder-to-order transitions, domain movements, quaternary and tertiary changes were observed, and the largest changes occurred in complexes with large interfaces.

Mass spectral characterization of a protein-nucleic acid photocrosslink.

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.

Thermodynamic Database for Protein-Nucleic Acid Interactions

Protein-nucleic acid interaction plays an essential role in the regulation of gene expression. Structural and binding information provides insight into the mechanism of protein-nucleic acid recognition. Although there are structural databases for protein-nucleic acid complexes, there exists no database for protein-nucleic acid binding. Thus, we have decided to develop the Thermodynamic Database for Protein-Nucleic Acid Interactions. It contains several important thermodynamic parameters, experimental conditions, structural information about proteins, nucleic acids and of the complex, and literature information. This database will be helpful for understanding the mechanism of proteinnucleic acid recognition. A WWW interface will help users to search the data based on various conditions with different sorting options for outputs.

Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: application to DNA opening by Eσ70 RNA polymerase

Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO4, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO·).To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO4 footprinting of the non-template strand of Eσ70 RNA polymerase-λPR promoter DNA complexes populated at binding equilibrium at 37°C and transiently populated at a fixed time after a temperature downshift from 37°C to 0°C. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RPo) both at 37°C and (transiently) after a downshift to 0°C; (ii) does opening of the start site region in RPo occur entirely in one mechanistic step at the λPR promoter and (iii) does the structure of RPo vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RPo) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RPo to free promoter DNA after a 37° to 0°C temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RPo.

Probing the Binding Region of the Single‐Stranded DNA‐Binding Domain of Rat DNA Polymerase β Using Nanosecond‐Pulse Laser‐Induced Cross‐Linking and Mass Spectrometry

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.

PQS: a protein quaternary structure file server

Part of the procedure outlined here uses a modification to the WHATIF program specially written for this purpose by R. Hooft at EMBL Heidelberg. J. Westbrook of the Nucleic Acid Data Base group at the University of Rutgers (http:// ndbserver.rutgers.edu) made available a machine-readable list of the quaternary states for all nucleic acid and protein–nucleic acid complexes held in the PDB that was used in the annotation and checking stage described here.

Activating Transcription Factor-2 Regulates Phosphoenolpyruvate Carboxykinase Transcription through a Stress-inducible Mitogen-activated Protein Kinase Pathway

Several protein-nucleic acid complexes are observed when nuclear extracts from hepatoma cells are assayed for binding to the cAMP response element found in the phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-C) promoter. Although cAMP response element-binding protein and CCAAT/enhancer binding proteins alpha and beta have been identified as liver factors that bind this motif, an uncharacterized, slower migrating complex was also observed. We identify activating transcription factor-2 (ATF-2) as the factor in this complex and show that ATF-2 stimulates expression from the PEPCK-C promoter. ATF-2 is a basic-leucine zipper transcription factor and a target for stress-activated protein kinases. We demonstrate that p38beta mitogen-activated protein (MAP) kinase augments ATF-2 transactivation activity on the PEPCK-C promoter, which is consistent with the interpretation that PEPCK-C promoter activity is maintained under stress through a p38 MAP kinase dependent pathway. In this regard, we show that treatment with sodium arsenite, a known activator of p38 MAP kinases, also stimulates expression from the PEPCK promoter. These results show that ATF-2 can stimulate transcription of the PEPCK-C promoter and support a role for stress inducible kinases in the maintenance of PEPCK-C expression.

NMR structure determination of proteins and protein complexes larger than 20 kDa

Recent advances in multidimensional nuclear magnetic resonance methodology to obtain 1H, 15N and 13C resonance assignments, interproton distance and torsion angle restraints, and restraints that characterize long-range order, coupled with new methods of structure refinement and novel methods for reducing linewidths, have permitted three-dimensional solution structures of single chain proteins in excess of 250 residues and multimeric proteins in excess of 40 kDa to be solved. These developments may permit the determination by nuclear magnetic resonance of macromolecular structures up to molecular weights in the 50–60 kDa range, thereby bringing into reach numerous systems of considerable biological interest, including a large variety of protein-protein and protein—nucleic acid complexes.

Plasmodesmata: Dynamics, Domains and Patterning

Although it has long been known that plasmodesmata establish cytoplasmic continuity between most cells within the body of the plant, it is only recently that these special structures have been viewed as dynamic intercellular organelles (Lucas and Wolf,Trends in Cell Biology3: 308–315, 1993) involved in the transport of macromolecules. Ultrastructural studies have provided important information on the formation of plasmodesmata during cytokinesis (primary plasmodesmata) and as post-cytokinetic events, where the new cytoplasmic bridges are inserted within the existing wall (secondary plasmodesmata). Modifications to plasmodesmal frequency, and presumably composition probably reflect developmental and physiological requirements for symplasmic continuity/communication. Pioneering studies on viral movement proteins provided the first direct experimental evidence that proteins and protein-nucleic acid complexes could traffic cell to cell, via plasmodesmata. This knowledge paved the way for the identification and characterization of endogenous proteins that also possess a similar capacity for cell-to-cell transport. Such proteins range from plant transcription factors, involved in orchestration of plant development, to proteins present in the phloem sap of angiosperms which are probably involved in maintenance of the enucleate sieve tube system. Information from viral infection studies and plant development support the concept that plasmodesmata play an essential role in the formation of developmental and physiological domains in which regulated trafficking of macromolecules establishes a non-cell-autonomous control network. Finally, the role of plasmodesmal trafficking of informational molecules is discussed in terms of providing a novel means for controlling cell differentiation. This concept is illustrated using current knowledge on root hair and trichome pattern formation.