Study Of Protein-nucleic Acid Interactions Research Articles

Optical tweezers in single-molecule biophysics.

Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein-nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth.

Efficient modification of \u03bb-DNA substrates for single-molecule studies

Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90–100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5′-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.

High-resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins.

Optical tweezers have become a powerful tool to investigate nucleic-acid processing proteins at the single-molecule level. Recent advances in this technique have now enabled measurements resolving the smallest units of molecular motion, on the scale of a single base pair of DNA. In parallel, new instrumentation combining optical traps with other functionalities have been developed, incorporating mechanical manipulation along orthogonal directions or fluorescence imaging capabilities. Here, we review these technical advances, their capabilities, and limitations, focusing on benchmark studies of protein-nucleic acid interactions they have enabled. We highlight recent work that combines several of these advances together and its application to nucleic-acid processing enzymes. Finally, we discuss future prospects for these exciting developments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 704-714, 2016.

Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

Single molecule studies of protein-nucleic acid interactions shed light on molecular mechanisms and kinetics involved in protein binding, translocation, and unwinding of DNA and RNA substrates. In this review, we provide an overview of a single molecule fluorescence method, termed "protein induced fluorescence enhancement" (PIFE). Unlike FRET where two dyes are required, PIFE employs a single dye attached to DNA or RNA to which an unlabeled protein is applied. We discuss both ensemble and single molecule studies in which PIFE was utilized.

Chapter 12 - DNA curtains: Novel tools for imaging protein–nucleic acid interactions at the single-molecule level

Interactions between proteins and nucleic acids are at the molecular foundations of most key biological processes, including DNA replication, genome maintenance, the regulation of gene expression, and chromosome segregation. A complete understanding of these types of biological processes requires tackling questions with a range of different techniques, such as genetics, cell biology, molecular biology, biochemistry, and structural biology. Here, we describe a novel experimental approach called "DNA curtains" that can be used to complement and extend these more traditional techniques by providing real-time information about protein-nucleic acid interactions at the level of single molecules. We describe general features of the DNA curtain technology and its application to the study of protein-nucleic acid interactions in vitro. We also discuss some future developments that will help address crucial challenges to the field of single-molecule biology.

Use of chromophoric ligands to visually screen co-crystals of putative protein-nucleic acid complexes.

Distinguishing between crystals of protein-nucleic acid complexes and those containing protein alone is a common problem in structural studies of protein-nucleic acid interactions. Currently, there are several methods available for detecting nucleic acid in crystals, including gel electrophoresis, SYBR Gold fluorescence dye staining, and methyl violet staining. However, they require either that the crystals be sacrificed or access to a fluorescence microscope. In this protocol, we describe an approach that allows direct visualization of either the presence or absence of oligonucleotides in crystals grown from solutions containing both protein and nucleic acid--labeling with the Cy5 dye. In addition to offering the advantage of being able to distinguish between crystals of complex and protein alone with the naked eye or a light microscope, crystals of covalently Cy5-labeled DNA can be directly used for X-ray diffraction data collection.

A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels.

DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous (32)P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.

Footprinting with exonuclease III.

AbstractWithin the last few years footprinting techniques have become increasingly important in the study of protein-nucleic acid interactions. This is partly the result of a fast-growing number of known nucleic acid-binding proteins but also because of an increase in the available probes that can be chosen in order to tackle a specific problem. There are two major groups of probes—the chemical probes and the enzymatic probes. The enzymatic probes, such as DNase I or exonuclease III, have the advantage of acting specifically on the DNA. Chemical probes are often less specific and may also react with the protein, possibly disturbing the correct interaction of protein with DNA. For the study of very fragile protein-DNA complexes, enzymatic probes are therefore often preferable.KeywordsAmmonium PersulfateSpeedVac ConcentratorAcrylamide SolutionEnzymatic ProbeFootprinting TechniqueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Identification of the amino acid residues of the amino terminus of vimentin responsible for DNA binding by enzymatic and chemical sequencing and analysis by MALDI-TOF.

The amino acid residues responsible for stable binding of nucleic acids by the intermediate filament (IF) subunit protein vimentin were identified by a combination of enyzmatic and chemical ladder sequencing of photo-cross-linked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28)(-)(35), vim(36)(-)(49), and vim(50)(-)(63)) were found to be cross-linked to oligo(dG.BrdU)(12). dG.3'-FITC. From a methodological standpoint, it was necessary to remove the bulk of the bound oligonucleotide by digestion with nuclease P1 to get reproducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the amino terminus of the peptide-oligonucleotide complexes with dimethylaminoazobenzene isothiocyanate dramatically improved the quality of the MALDI-TOF spectra obtained, particularly for the vim(28)(-)(35) peptide. A single Tyr residue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequivocally demonstrated to be the unique site of cross-linking in each peptide. These three Tyr residues are contained within the two beta-ladder DNA-binding wings proposed for the middle of the vimentin non-alpha-helical head domain. The experimental approach described should be generally applicable to the study of protein-nucleic acid interactions and is currently being employed to characterize the DNA-binding sites of several other IF subunit proteins.

Raman difference studies of protein-nucleic acid interactions

Raman difference spectroscopy combined with isotope editing procedures were employed to obtain the Raman spectra of phosphates, nucleotides, nucleic acids and their fragments when bound to several proteins. The vibrational spectra were analyzed using a variety of empirical and ab initio methods to relate the vibrational wavenumbers to hydrogen bonding strengths, bond orders, bond lengths and geometry and to establish how the proteins bring about these interactions. Some recent results obtained using these methods are reviewed. Copyright © 1999 John Wiley & Sons, Ltd.

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

Oligonucleotides of a novel type containing 2′- O-β-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein-nucleic acid interactions.

Footprinting with exonuclease III.

Within the last few years footprinting techniques have become increasingly important in the study of protein-nucleic acid interactions. This is partly the result of a fast-growing number of known nucleic acid binding proteins but also because of an increase in the available probes that can be chosen in order to tackle a specific problem. There are two major groups of probes-the chemical probes and the enzymatic probes. These enzymatic probes, such as DNase I or exonuclease III, have the advantage of acting specifically at the DNA. Chemical probes are often less specific and react also with the protein. This can disturb the correct interaction of protein and DNA. For the study of very fragile protein-DNA complexes, therefore, enzymatic probes are often preferable.

Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.

The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, highly fluorescent complexes with double-stranded DNA that can be detected in gels by a laser-excited, confocal, fluorescence scanning system with a sensitivity higher than that attainable with radioisotopic labeling. We describe here the use of these dyes in a gel-mobility assay to detect complexes of a truncation of the Kluyveromyces lactis heat shock transcription factor, containing the trimerization and DNA-binding domains (HSFDT), with target DNA. At an appropriate molar DNA base pair to dye ratio, the labeling of a DNA fragment with dimeric dye did not affect the binding to HSFDT. The detection of the fluorescent-dye labeled HSFDT-DNA complexes with the laser scanner achieves a spatial resolution far superior to that of conventional autoradiography and permits analysis of multimer protein-DNA complexes that are not resolved by traditional detection methods. We have used this technique to demonstrate that HSF forms multimeric complexes on DNA by addition of trimeric units. The latter conclusion is based on an analysis of the mobilities of the multiple HSFDT-DNA complexes and on a two-color mobility-shift fluorescence assay that uses a mutant of HSFDT engineered for site-specific labeling with fluorescein and target DNA labeled with an "energy transfer" dye, thiazole orange-thiazole blue heterodimer.

DNA-protein interactions, structural studies of protein-nucleic acid interactions

DNA-protein interactions, structural studies of protein-nucleic acid interactions

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

Energy transfer: A general method for the study of protein-nucleic acid interactions

A novel fluorescence method has been developed which should have general applicability for the study of protein-nucleic acid interactions. The association between proteins covalently labeled with dansyl chloride and DNA noncovalently labeled with acridine orange can be studied by monitoring the transfer of fluorescent energy from the donor (the dansyl label) to the acceptor (acridine orange). This method has been illustrated in model fluorescence studies which yield estimates of the combining ratio of histone VIIIS (Sigma, arginine rich) with DNA.

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH. At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants ( K D). Values of K D ranging from 10 −6 m at pH 2.7, 0 °C, to 2 × 10 −10 m at pH 7.0, 0 °C, are reported here. Our value for K D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971). At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation. Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.

Nuclear magnetic resonance studies of protein-nucleic acid interactions: II. The E. coli tRNA Glu complex with glutamyl-tRNA synthetase

We have observed the 300 MHz high-resolution proton nuclear magnetic resonance spectrum of the Escherichia coli tRNA Glu complex with the glutamyl-tRNA synthetase. The observations are fitted very well by a computer-simulated spectrum of the E. coli tRNA Glu itself, with the individual resonances broadened from 45 Hz to 150 Hz because of the increased molecular weight. This indicates that no helical arms open upon complex formation, nor is there evidence for any additional Watson-Crick base-pairs in the complex.