Visualizing nucleic acids and their complexes using electron microscopy

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Visualizing nucleic acids and their complexes using electron microscopy Jack D Griffith*, Suman Lee and Yuh-Hwa Wang Microscopic visualization of nucleic acid-protein complexes provides a means of obtaining structural information that is difficult to obtain in any other way, and of verifying conclusions derived from other approaches. The polymorphic, flexible, and irregular nature of these complexes presents particular problems in their analysis.

Addresses Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA *e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:362-366

http://biomednet.com/elecref/Og59440XO0700362 © Current Biology Ltd ISSN 0959-440X Abbreviations ATM atomic force microscopy bp base pair EM electron microscopy ESI electron spectroscopic imaging kb kilobase STEM scanningTEM TBP TATA box binding protein TEM transmissionEM

Introduction As modern molecular biology moves toward more complex muhimolecular systems, the need for structural information about nucleic acid-protein assemblies grows. This information can be obtained from a variety of physical methods, but one domain of structural biology remains uniquely approachable via microscopy--the visualization of nucleic acid-protein complexes involving DNAs that are frequently thousands of base pairs in size. The need for reliable methods for analyzing such structures has been underscored by an interest in how enhancers act at a distance along the DNA. Microscopy provides a means of approaching these i s s u e s - - b u t only if reasonable criteria for analysis are applied. We will discuss such criteria and review the advances in several select areas that illustrate recent accomplishments. Criteria for the use of electron microscopy to study nucleic acid-protein complexes Most complexes of DNA or RNA with proteins (other than structures such as ribozymes) are inherently flexible, large, irregular, and polymorphic--features that preclude microscopic methods that produce a single 'structure' by averaging many images or by the generation and analysis of 2D crystals. The typical complex of DNA and protein that is the topic of this review might consist of a 3kb DNA containing a pair of protein-binding sites each

separated by 1 kb. In the analysis of such complexes, the structures formed at the two sites by the site-specific binding proteins and any possible looping between the two sites would be examined. For such studies, four general criteria provide valuable guidelines. Once these have been satisfied, additional analysis using other electron microscopy (EM) approaches will be on a stronger footing. The basic conclusions of the study must be apparent by direct observation of the samples using EM without the imposition of any image enhancement or averaging process While much useful information can be derived from averaging and enhancement methods, it is essential that the images of the complexes be of a clarity such that if the general nature of the sample were to be explained to a naive observer at the EM, he or she would come to the same conclusion as the person preparing the samples. This requirement derives from the polymorphic nature of such samples. If the sample is not easily observed, attention may become fixed on nonrepresentativc structures, and it becomes too easy to select just those images that fit a particular model. It must be possible to collect many hundred examples and to place them into distinct structural classes Analysis of polymorphic samples demands a statistical analysis of a large data set. Nucleic acid-protein complexes frequently contain partially assembled structures. Further, the complexes normally adsorb to the planar supports in several different orientations. Thus, it is seldom that any singular example will be fully representative of the spectrum of structures, and a representative description requires a detailing of the different forms present and their statistical frequency. The sample preparation process itself must be reliable and relatively easily carried out The most useful molecular biology experiments utilizing microscopy are usually the ones in which changes seen by EM are correlated with changes in the sample that occur with time or with alterations of reaction conditions. For such work to be accomplished, many samples need to be prepared frequently over a short period of time. Sample preparation methods that are slow or cumbersome preclude such correlations. The conclusions of the study must be unaffected by (transparent to) possible artifacts of sample preparation Each EM preparative method has inherent weaknesses such as exposure to fixatives, high salt (as in negative staining), or adsorption of the sample to a flat supporting

Visualizing nucleic acids and their complexes Griffith, Lee and Wang

surface. Whereas such concerns may impose certain limitations on conclusions derived about the fine structure of the complexes themselves, the fundamental conclusions must not be compromised by questions of sample preparation. For example, if the study describes the localization of a binding site for a protein along a DNA, the precise structure of the protein bound to the DNA may or may not be affected by fixation with glutaraldehyde, but it would be difficult to argue that mapping 90% of the protein molecules to a single site along the DNA was an artifact of fixation. Some of the most commonly used methods of fixation have been reviewed elsewhere [1-5]. Nucleic acids and their protein complexes The study of chromatin structure remains one of the more mature fields of microscopy in which the different EM methods are combined with gel electrophoresis, chemical probing, and in vitro reconstitution techniques to derive structural information relevant to basic biological questions. Rotary shadowcasting, cryoEM, and scanning transmission electron microscopy (STEM) have been applied to issues concerning how DNA wraps around the nucleosome core [61, the role of the linker histones [7], the fate of nucleosomes during transcription [8°'], and the presence of DNA elements that strongly position or exclude nucleosomes. The latter issue is of particular interest since EM was used to show that repeating microsatetlite DNAs whose expansion triggers several human genetic diseases will in one case (Myotonic Dystrophy) strongly position nucleosomes and in another case (Fragile X syndrome) strongly exclude nucleosomes [9,10"°]. Although determining the fine structure of DNA and RNA has generally remained the domain of X-ray and NMR methods, one new approach--helix extension microscopy--has been developed [11 °°] that provides

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a means of probing the 3D folding of complex RNA molecules such as ribozymes. In this approach, individual RNA helices are extended in vitro to be long enough for transmission electron microscopy (TEM) visualization. The angles between two extended helices are measured pairwisc for several sets of helices to produce a 3D ribozyme structure. Although the RNA constructs involved are not trivial to prepare, the microscopic analysis is straightfo~,ard and provides information difficult to obtain by other approaches.

An excellent example of the unique power of EM to provide information difficult to obtain by conventional biochemical means can be found in a pair of studies in which the binding of the human tumor suppressor protein p53 [12] and the yeast mismatch repair protein MSH2 [13] to Holliday junctions was examined. As shown in Figure 1, EM determined that the protein is localized to the junction in the DNA and not along the arms or at the ends of the DNA--information that can not be obtained using gel shift or filter binding assays. Advances in understanding the structure of splicing complexes have been made using STEM and targeted biotinylated antisense oligonucleotides. Streptavadin-gold has been used to unambiguously identify the location of RNA in the splicing complexes [14]. In a study of UBF protein bound to DNA, the structures have been imaged by both positive staining and electron spectroscopic imaging (ESI), in which the system is tuned to image phosphorus on unstained samples [15°°], and these data are augmented with molecular modeling to present a high resolution structure. Conventional shadowcasting and negative staining continue to provide information about RNA-protein complexes, as in the recent study of TRAP protein [16]. In a detailed study of the eukaryotic single-stranded DNA-binding protein hRPA, Blackwell et

Figure 1

Visualization of p53 protein bound to Holliday junctions, Human p53 protein produced in insect cells is incubated with artificial Holliday junction DNAs. These DNAs are generated by annealing - 5 5 0 bp duplex arms onto a four-way junction (containing 25 bp arms) formed by the annealing of oligonucleotides synthesized to produce a frozen Holliday junction. The complexes are prepared for EM by fixation with glutaraldehyde, adsorption to thin carbon foils, dehydration, and rotary shadowcasting with tungsten in a very high vacuum. The p53 protein can be seen located at the junction as opposed to along the length of the arms or at their ends, a finding that is difficult to demonstrate using other methodologies. The images are shown in reverse contrast.

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al. [17] have utilized STEM to present a detailed analysis

of the site size and configuration of hRPA bound to single-stranded DNA.

DNA bending Microscopy has contributed much to our understanding of how nucleic acids are bent by sequence motifs or by proteins. The first direct demonstration that phased tracts of As bend D N A was obtained by EM [18]. Revet and coworkers [5] have utilized EM to generate a detailed bending map for pBR322 plasmid DNA, which has provided an extremely valuable database for theoretical models of DNA bending. Recently, electrophoretic mobility shift methods have been devised for measuring bending. EM analysis, however, can determine both a mean angle of bending for the population, and information about the distribution of bending angles, data that are difficult to derive from gel analysis. Furthermore, for DNAs bent by large protein complexes, it is unlikely that the contribution of the protein complex to the electrophoretic migration can be discounted, and thus a direct EM analysis would be more reliable. In a recent EM study of the bending of DNA by yeast TATA box binding (TBP) protein, a series of EM approaches have been applied [19°']. In an earlier analysis of the bending of DNA by the uvrA and uvrB repair proteins, it was found that uvrB bends DNA severely [20], and recently this study has been extended to show that a single amino acid mutation in uvrB eliminated both bending and its ability to facilitate repair [21]. DNA looping The long range structural sculpting of DNA into functional domains or loops was first demonstrated by EM for a 60bp loop induced by the lambda repressor [22]. Since then, looping of DNA has become a common explanation for action observed at a distance along DNA. Looping remains difficult to demonstrate biochemically but is easily seen by EM. Indeed, this is one of the simplest, relatively artifact-free determinations. If the length of the DNA loop is greater than - 5 0 0 b p , and the loops can be stabilized by fixation, then simple cytochrome c surface spreading and conventional EM can be applied [3], and gold tagged antibodies may be used to identify the proteins at the base of the loop. As detailed in Thresher and Griffith [3], cytochrome c spreading--a method in which the sample is spread on an air-water interface in a film of denatured cytochtome c--provides a powerful albeit low resolution means of visualizing DNA, and, in some cases, protein bound to DNA. Rotary shadowcasting of looped DNA-protein complexes has been utilized by most investigators, and this method easily satisfies the criteria presented above. Earlier concerns over artifactual loops created by glutaraldehyde fixation have not been borne out, and the frequent use of fixation in gel retardation analysis of DNA-protein complexes has made investigators more comfortable with this approach. In one looping study, a series of loops has been observed

to form in the upstream control region of a sea urchin gene, and different loops have been postulated to function during different stages of differentiation [23]. Looping of DNA between p53 transcriptional activator sites has been described [24], and several studies have reported looping of DNA at origins of replication including that of the Epstein-Barr virus [25,26], one in Chinese hamster ovary cells [27], and the otiS origin in Herpes Simplex Virus [28°]. In the latter case, the loops were found to grow in size with ATP hydrolysis. Because looped molecules may represent a minor fraction of the total DNA population, EM provides a unique means of studying this central feature of active DNA. EM mass analysis

Determining the mass of a protein complex assembled onto DNA or RNA using indirect biochemical approaches is difficult but has been accomplished with relative ease using EM. Such analyses have been taken to sophisticated levels using dedicated STEM instruments such as the one at the Brookhaven National Laboratory constructed by Wall and coworkers. They have measured masses to within a standard deviation of 5% or less and have done so over a large molecular weight range including structures of several megaDaltons [29,30]. Often, 1000 particles are averaged for each determination, and Tumminia et al. [29] have found that the correlation between theoretical and measured values for ribosomal subunits is striking. Frequently, for studies of DNA-protein complexes, the issue is whether a protein binds to DNA as a monomer, dimer or higher oligomer. If the sample is imaged by rotary shadowcasting or negative staining, the inclusion of proteins of known mass makes it possible to determine the degree of oligomerization using conventional EM. Using a single-stranded DNA-binding protein (SSB) as a marker, the mass of yeast TBP (monomer mass 28 kDa) bound to a TATA box was measured to be 56+5 kDa, revealing dimerization of TBP [18]. Such measurements work well as long as the protein complexes are in the range of 50 to several hundred kiloDaltons, the protein particle is similar in size and shape to the internal standard, and the D N A makes a relatively minor contribution to the mass. CryoEM

Three general approaches have been taken that utilize cryofixation of DNA-protein complexes. Heuser [31] has adsorbed samples to mica chips which are then slam frozen in liquid helium. The sample is exposed by deep etching and imaged by metal shadowcasting. The images of recA protein-DNA complexes produced using this method are striking [32]. In a somewhat different approach, Bortner and Griffith [33] have adsorbed samples to typical EM supports and frozen the sample in liquid ethane slush. This is followed by a freeze-drying and rotary shadowcasting process carried out in an ultrahigh vacuum system [33]. Cryoimaging of DNA and DNA-protein complexes frozen in ice has been developed and perfected by DuBochet and coworkers [34]. Their clear images show

Visualizing nucleic acids and their complexes Griffith, Lee and Wang

linear and supercoiled DNA, and DNA bound by RNA polymerase [34]. Careful analysis of supercoiled DNA in ice has provided a new generation of understanding of the shape, writhe, and persistence length of supercoiled DNA [35,36]. We expect that as the methods and equipment become simplified, this technology will see wider use and greater input into molecular biology, as it offers a 3D view of hydrated macromolecules that cannot be obtained by any other means.

Scanning tip microscopies While scanning tip microscopies (atomic force, scanning force, scanning tunneling) are not formally EM, they offer potential new approaches for imaging nucleic acid-protein complexes, and it is worth comparing their advantages and disadvantages to classic T E M / S T E M . The advantages of EMs include, first, their ability to rapidly magnify fields over a range from tenfold to nearly 1 millionfold, and, second, and perhaps most important, that the image formed in the EM presents an image of the sample i t s e l f - - f o r example, each tiny speck in the image is a real particle of metal in the sample. In contrast, the magnification range for the scanning tip microscopies is not as easily varied, and the images produced reflect in part the structure of the scanning tips themselves, and thus much more care is needed in the interpretation of the images. T h e greatest potential advantage of the scanning tip microscopes is the possibility of imaging samples in physiological buffers. Great strides have been taken in these new methods. DNA has been imaged in buffer as well as in a dehydrated form [37"',38], and the images are convincing. Possibly, the most interesting applications have been in the area of chromosome structure [39-41]. Indeed, it may turn out that while the classic microscopies, together with cryoEM of samples in ice, will remain the most productive means of imaging DNA and DNA-protein complexes, the scanning tip microscopies will find their most important applications in the area of imaging larger structures such as chromosomes, sperm, and cellular substructures in physiological buffers.

Conclusions Microscopy at the molecular level will continue to make critical contributions to our understanding of molecular biology as we move toward understanding macromolecular machines and assemblies. In the field of nucleic acid-protein complexes, the inherently polymorphic, flexible, and irregular nature of large assemblies of protein bound along DNA or RNA imposes limitations on the approaches that can be used and demands an approach that is based on the statistical analysis of many complexes. Laboratories taking such an approach have made significant findings in the area of DNA and RNA structure, DNA bending and looping, and chromatin structure. In the future, we expect that greater application of cryoimaging in ice will be seen. Scanning tip microscopies continue to offer potential, but possibly more

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for subcellular structures imaged in physiological buffer than for nucleic acid structures.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •.

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