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a. For the in vitro assembly of 40 S monoparticles, the added RNA or single-stranded DNA should be between 685 and 726 nt in length or integral multiples of 700 nt for the assembly of dimers and oligomeric complexes. Other RNA lengths will support the assembly of nonstoichiometric complexes which sediment through the gradients. The core proteins of 40 S hnRNP do not assemble in a sequence-dependent manner in vitro, and nucleotide triphosphates are not required as an energy source. b. For correct particle reassembly, the exogenous RNA should be added at 0.7 times the measured OD of the pooled and dialyzed sample. In other words, add 0 . 7 0 D units (28/xg) of exogenous RNA per OD unit of isolated hnRNP. Correct particle assembly will occur over the range 10-20 : I for protein to RNA. Reactant concentration has no significant affect on particle assembly.
[25] P h y s i c a l M e t h o d s for C h a r a c t e r i z a t i o n o f H e t e r o g e n e o u s Nuclear Ribonucleoprotein Complexes
By JERZY T. SCHONEICH a n d JOHN O. THOMAS The major components of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are transcripts of RNA polymerase II, a rather complex set of proteins, and small nuclear (sn) RNAs2 ,2 The sizes of the complexes and their shapes as judged from electron micrographs depend to a large degree on the method used for their preparation. The largest complexes observed sediment at about 200 S. Complexes of this size can be obtained by sonicating nuclei in the presence of RNase inhibitors. 3 In the presence of an RNase activity, either endogenous or an added nuclease, these large complexes are degraded, yielding smaller complexes that sediment in a rather broad band at about 40 S. These complexes contain RNA fragments that range in size from about I00 to about 1000 nucleotides (nt). 4 When viewed by electron microscopy, these particles have an S. Y. Chung and J. Wooley, Proteins: Struct. Funct. Genet. 1, 195 (1986). 2 G. Dreyfuss, Annu. Reo. Cell Biol. 2, 459 (1986). a R. Sperling, P. Spann, D. Often, and J. Sperling, Proc. Natl. Acad. Sci. U.S.A. 83, 6721 (1986). 4 y. D. Choi and G. Dreyfuss, Proc. Natl. Acad. Sci. U.S.A. 81, 7471 (1984).
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average diameter of about 20--25 nm, although they are rather heterogeneous in size and shape. 5 Because of the heterogeneous nature of hnRNP complexes, it is difficult to apply directly the physical methods traditionally used for elucidating the structures of macromolecules. One alternative is to study complexes reconstituted with nucleic acids of defined sizes and to examine the properties of the reconstituted complexes as a function of the size of nucleic acid that is in the complex. Several approaches for doing this are described here.
Principles o f Analysis The principle of this approach is to examine the structures formed between proteins and nucleic acids of defined lengths. Changes in the size and shape of the complexes as the lengths of the nucleic acids increase can be related to a stepwise buildup of the complexes and will reflect their structure and the way in which they are assembled. Depending on the size of nucleic acid that is chosen and the method that is used for analysis, several questions can be addressed. By studying the binding to a series of small oligonucleotides of increasing sizes, one can determine binding site sizes and binding constants and also measure cooperative interactions between neighboring proteins. 6 Also, by examining the effects of solvents on these parameters, some information about the nature of the binding forces (such as the number of ionic interactions) that are involved can be obtained. By using longer nucleic acids as substrates, higher order structures that involve several or many proteins can be examined. In particular, the size and composition of cooperative units can be determined by sedimentation and electrophoresis, and the molecular organization of the complexes can be observed by electron microscopy.
Preparation o f Defined-Length Nucleic Acids RNAs of defined lengths and sequences can be easily obtained by in vitro transcription. A number of procedures using various promoters have been described, 7,8 and several kits are commercially available. For some analyses a heterogeneous mixture of RNAs of various sizes is desirable. This can be produced either by limited alkaline hydrolysis of a transcript (see below) or by synthesizing the RNA in the presence of a limiting 5j. Wooley,s. Y. Chung,J. Wall, and W. LeStourgeon,Biophys. J. 49, 17 (1986). 6j. D. McGheeand P. H. Von Hippel, J. Mol. Biol. 86, 469 (1974). 7 D. A. Melton,P. A. Krieg,M. R. Rebagliati,T. Maniatis,K. Zinn,and M. Green,Nucleic Acids Res. 12, 7035 (1984). 8 E. T, Schenborn and R. C. Mierendorf, Jr., Nucleic Acids Res. 13, 6223 (1985).
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amount of nucleotide, which results in premature terminations and a distribution of RNA sizes. Although the hnRNP proteins are found in association with hnRNA, they also appear to be capable of binding single-stranded DNA. 9-14Large quantities of defined sizes of single-stranded DNAs can be obtained by denaturing DNA fragments generated by restriction enzymes. These can be end-labeled by the Klenow fragment of DNA polymerase 15 prior to denaturation, or they can be labeled with T4 polynucleotide kinase after removal of the 5'-phosphate by phosphatase. 15The DNA can be rendered single stranded by dissolving it in 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, heating it in a boiling water bath, and quickly cooling it on ice. Homopolymers are useful for reconstitution because they provide simplified model systems that are homogeneous in composition and secondary structure. Homopolymers, which are typically several hundred nucleotides long, can be obtained from a number of commercial sources. Commercial poly(A) is usually longer than poly(U) or poly(C). Shorter homopolymers can be prepared by limited alkaline hydrolysis 16 (for deoxyhomopolymers, a limited digestion with micrococcal nuclease can be used), which leaves a free 5'-hydroxyl that can be conveniently labeled using T4 polynucleotide kinase. To obtain defined sizes of homopolymers, a partially hydrolyzed mixture can be separated by electrophoresing through a polyacrylamide gel, cutting the gel, and eluting the fragments of the desired sizes. To elute the fragments, the end of a plastic micropipet tip is plugged with a small amount of siliconized glass wool, and the end is sealed. The gel piece is placed in the pipet tip and crushed with a glass rod. After crushing, 0.5 ml of 0.5 M ammonium acetate, 1 mM EDTA is added, and the RNA is allowed to elute overnight. The end of the tip is then cut, the solution is drained, and the RNA is precipitated with 2.5 volumes of ethanol. For larger fragments, electroelution also works well. To produce a mixture of RNA fragments by alkaline hydrolysis,16 the RNA or homopolymer in 50 mM sodium carbonate buffer, pH 9.0, is 9 j. M. Pullman and T. E. Martin, J. Cell Biol. 97, 99 (1983). ~0H.-E. Wilk, G. Angeli, and K. P. Sch~fer, Biochemistry 22, 4592 (1983). i1 L. Nowak, D. K. Marvil, J. O. Thomas, M. Boublik, and W. Szer, J. Biol. Chem. 255, 6473 (1980). lz Raziuddin, J. O. Thomas, and W. Szer, Nucleic Acids Res. 10, 7777 (1982). 13 A. Kumar, K. R. Williams, and W. Szer, J. Biol. Chem. 261, 11266 (1986). 14A. Kumar, H. Sierakowska, and W. Szer, J. Biol. Chem. 262, 17126 (1987). 15 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 16 H. Donis-Keller, A. M. Maxam, and W. Gilbert, Nucleic Acids Res. 4, 2527 (1977).
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heated to 65 ° in a microcentrifuge tube. After 3, 10, and 25 min, samples are placed on ice and neutralized with 1/10 volume of 2.5 M sodium acetate, 10 mM MgCIE, pH 5.2. Because the rate of degradation is very sensitive to the exact conditions used, particularly the pH, it is necessary to check the extent of hydrolysis of each sample. This is done by 5'-endlabeling a small portion and analyzing it by polyacrylamide gel electrophoresis and autoradiography. Reconstitution with Total h n R N P Proteins
Methods for forming reconstituted hnRNP complexes using exogenously added nucleic acids have been described. 9a° All of these methods suffer from the lack of a suitable structural or functional assay for "native" hnRNP complexes, so it is never certain how faithfully the reconstituted particles reflect the structure of the complexes that are formed in vivo. Nonetheless, in vitro reconstitution studies do provide a handle for investigating the RNA-protein and protein-protein interactions that can occur during the formation of the structures. This is a useful and perhaps essential framework for examining these rather multifarious particles. The simplest method for reconstitution is to digest the endogenous RNA and then add the desired nucleic acid in its place. The proteins associate spontaneously with the added nucleic acid. The hnRNP complexes [1 mg/ml protein in l0 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1), 50 mM NaC1, 1 mM CaC12, 1 mM dithiothreitol (pH 7.6)] are digested with 2.5 units/ml micrococcal nuclease. After 10 rain at 37°, the nuclease is inactivated by adding EDTA to 5 mM. The exogenous RNA is then added to give a protein to nucleotide molar ratio between I : 6 and 1 : 12. The composition of the buffer is not particularly critical, although elevated salt concentrations tend to favor the dissociation of most nucleic acid-protein complexes and very low concentrations of salt may lead to the precipitation of hnRNP proteins. Magnesium does not appear to have much influence on the formation of the complexes. Dithiothreitol (DTT) is routinely included since the free proteins tend to precipitate in its absence. 13,14 For the reconstitution of complexes with poly(A), the endogenous RNA can be digested with RNase A. Since poly(A) is not degraded by RNase A, the complete inhibition of the enzyme is not a consideration as it is with micrococcal nuclease. Reconstitution with Individual Proteins
A few hnRNP proteins have been purified and partially characterized. n-14,17 They appear to share the ability to bind to RNA (and single17 F. Cobianchi, R. L. Karpel, K. R. Williams, V. Notario, and S. H. Wilson, J. Biol. Chem. 263, 1063 (1988).
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stranded DNA) and form condensed nucleic acid-protein complexes. 12,1a,19 The best characterized complexes are those formed with HD40, the major hnRNP protein of Artemia salina. HD40 condenses nucleic acids into helices that are only marginally stable; on average, the helix formed by the nucleoprotein complex is only about 2] turns. 18 The structure of the hnRNP complexes, and the way in which the nucleic acids are condensed, can be examined by observing the particles formed when the proteins associate with nucleic acids of increasing lengths. The effect of varying the protein to nucleotide ratio and the salt conditions used for reconstitution can also be examined to give additional information about the structure of the complexes and the forces involved in their formation. Homopolymers of defined sizes are good substrates for these studies since complications arising from sequence specificities of the proteins and differences in secondary structures of the nucleic acids can be minimized. Although the complexes can be observed with almost any physical technique, electron microscopy, electrophoresis, and hydrodynamic methods are particularly informative. Typically, the nucleic acid-protein complexes are formed simply by mixing the protein and nucleic acid. It is important to keep the protein concentration as high as possible to assure that the complex is fully saturated (the complexes that have been examined have intrinsic association constants of about 105-106 M - l ) . llA7 Following the formation of the complex, it can be analyzed as described below.
Analysis of Reconstituted Complexes Sedimentation. As mentioned above, sedimentation has been widely used for the isolation and characterization of hnRNP complexes. Sedimentation can also be used in conjunction with electrophoresis for the analysis of hnRNP complexes reconstituted with either defined-length RNAs or with a heterogeneous mixture of RNAs of various sizes. Following reconstitution (see above), the mixture is layered on top of a 10-30% (w/w) sucrose gradient made with a buffer containing 10 mM Tris-HC1, 25 mM NaC1, 5 mM EDTA, 1 mM DTT, pH 7.6, and centrifuged. For sucrose gradients made on a percent (w/w) basis, a good approximation of the sedimentation coefficient as a function of the position in the gradient can be easily calculated, 2° although a more exact determination should be made by comparing the rate of sedimentation with ribosomal markers. For analytical purposes, centrifugation in a Beckman SW 50.1 rotor is ~8j. O. Thomas, S. K. Glowacka, and W. Szer, J. Mol. Biol. 171, 439 (1983). ~9j. T. Sch6neich and J. O. Thomas, unpublished observations (1988). 2o C. R. McEwen Anal. Biochem. 20, 114 (1967).
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convenient. After a 100-min centrifugation at 5° at 49,000 rpm, 60 S material should be located near the middle of the gradient. Following fractionation, the gradients can be analyzed by several methods. The simplest is to plot the distribution of radioactivity through the gradient. More information can be obtained, particularly if heterogeneous RNA is used, if the RNA is isolated from each fraction by phenol extraction and ethanol precipitation and then analyzed by electrophoresis. Separation of the RNA on 3.5% polyacrylamide-8 M urea gels is convenient for resolving RNAs between 0. l and 2 kilobases (kb) in size. For smaller RNAs, the concentration of acrylamide can be increased up to 20%. For larger RNAs, agarose-formaldehyde gels zl should be used. After separating the RNAs on gels, the size distribution of the RNAs in each fraction can be quantitated by densitometry of autoradiograms. From one experiment in which a broad size range of RNAs is used, the sedimentation behavior of complexes incorporating this population of RNAs can be determined. This enables a direct comparison to be made between complexes formed with RNAs of many different sizes under the exact same conditions. In addition to an analysis of the RNAs in each fraction of the gradient, the protein composition of each fraction can be determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. 22 For detection, the proteins can be stained either with Coomassie brilliant blue or w i t h silver. 23 The silver stain is much more sensitive, but it should be noted that with silver the major hnRNP proteins are less intensely stained relative to the other protein components of the hnRNP complexes than they are with Coomassie brilliant blue. A serious problem with examining the proteins present in complexes separated by sedimentation through sucrose gradients is that the hnRNP proteins readily aggregate in the absence of nucleic acid, particularly in low-salt buffers in the absence of DTT. 13,14This makes it difficult to distinguish between nucleic acid-protein complexes and aggregates of free proteins that might cosediment with the complexes. Analytical ultracentrifugation is useful for examining the interactions between purified hnRNP proteins and nucleic acids. From sedimentation equilibrium studies, the molecular weights of complexes formed with different sizes of nucleic acids can be determined, and from the change in molecular weight as a function of nucleic acid size, the excluded binding site size can be calculated.~8 The molecular weights can also be used in conjunction with sedimentation coefficients, determined by sedimenta21 H. Lehrach, D. Diamond, J. M. Wozney, and H. Boedtker, Biochemistry 16, 4743 (1977). 2~ U. K. Laemmli, Nature (London) 227, 680 (1970). 23 C. R. Merril, D. Goldman, S, A. Sedman, and M. H. Ebert, Science 211, 1437 (1981).
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tion velocity experiments using the same samples, to estimate frictional coefficients and axial ratios of the complexes.la Electrophoresis on Nondenaturing Gels. Electrophoresis through nondenaturing agarose gels provides an alternative to sedimentation for the separation of reconstituted hnRNP complexes. A major advantage of electrophoresis over sedimentation is that there is a clear separation of the RNA-protein complexes from free proteins and from unbound RNAs. This enables one to characterize both the RNA and protein components of the complexes that are formed. Reconstituted complexes [in 20 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES), 50 mM NaC1, 5% (v/v) glycerol, 1 mM DTT, 0.001% bromphenol blue (pH 7.6)] are loaded onto a 1% agarose gel [in 20 mM HEPES, 25 mM NaCl, 5% (v/v) glycerol, 1 mM DTT (pH 7.6)]. Electrophoresis is conducted in the cold at 3.5 V/cm until the dye front has migrated about 80% of the length of the gel. The running buffer [50 mM Tris-HC1, 25 mM NaC1, 1 mM DTT (pH 7.6)] should be continuously recirculated. In nondenaturing gels, the free hnRNP proteins remain near the origin. Free nucleic acids and nucleic acid-protein complexes move toward the positive electrode, with the free nucleic acids migrating much faster than nucleic acid-protein complexes. 19Separation of the complexes is based on a combination of size, shape, and net charge. The distribution of the RNA and protein components throughout the nondenaturing gel can be analyzed on second dimension gels: a polyacrylamide gel containing 8 M urea for the analysis of RNA or an SDS-polyacrylamide gel for the analysis of proteins. A disadvantage of using electrophoresis for the resolution of the complexes is the fact that one cannot a priori determine the relationship between sedimentation coefficients and mobilities on gels. Rather, it is necessary to correlate empirically the electrophoretic mobilities of the complexes with their sedimentation coefficients. Electron Microscopy. Electron microscopy is potentially an extremely powerful tool for examining the structure of macromolecular complexes. However, since the hnRNP complexes are rather heterogeneous, the value of electron microscopy has been limited. The problem of sample heterogeneity can be reduced by examining complexes reconstituted with defined sizes of oligonucleotides. This approach can be particularly powerful if the morphology of the complex is examined as a function of the size of the nucleic acid used for reconstitution. For electron microscopy, complexes are reconstituted with the desired nucleic acid as described above. Complexes containing nucleic acids longer than 100 nt can be easily visualized, but structures containing fewer than 50 nt are difficult to distinguish reliably from background
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debris. Complexes reconstituted from crude hnRNP proteins are also difficult to analyze because of a large background of proteins and protein aggregates. In order to preserve the structure of the complexes during the subsequent processing steps, it may be desirable to fix them by adding 1/20 volume of 8% glutaraldehyde to the reconstituted complexes.~8 On the other hand, the presence of the fixative might alter the structure of the complexes and may also lead to the formation of protein aggregates if fixation is excessive. Because of these problems, it is best to examine and compare both fixed and unfixed complexes. In our experience, there is usually little difference. Specimens can be mounted either by spraying them onto a mica sheet or carbon film or by direct adsorption onto a carbon film. Adsorption is much easier. For adsorption, thin carbon films on 400-mesh copper electron microscope grids 24 are used. The carbon-coated grids are generally hydrophobic and do not interact with the nucleic acid-protein complexes. There are several methods for making the grids hydrophilic. The easiest and most satisfactory method for nucleic acids coated with proteins is to activate the grids by placing them in a glow discharge. 25 We generate a glow discharge by passing a 55 mA current through a 80-120 mTorr vacuum. The grids should be kept in the discharge for about 8 min. Insufficient activation will not render the grids hydrophilic, and overactivation will result in increased background contamination. Consequently, it is best to determine empirically the optimal conditions for each particular apparatus. The activated grids should be used the same day. The nucleoprotein complexes are mounted on the activated grids simply by placing a 5- to 10-/xl drop of sample on the grids and allowing it to adsorb for about 30 sec. The amount of sample that is adsorbed onto the grid is dependent on the adsorption time, so dilute samples may require longer times. For negative staining, the droplet is washed off of the grid with several drops of 10 mM Tris-HCl, pH 7.5, applied rapidly with a Pasteur pipet. This is followed by several drops of stain. The stain is then immediately removed by touching a piece of paper towel or filter paper to the edge of the grid. When done properly, a thin film of stain will remain on the grid. This will quickly dry in the air, and the grids can be observed immediately. A number of stains are commonly used for the negative contrasting of macromolecules. 24,26 In our experience, the stain which 24 R. H. Haschemeyer and R. J. Myers, in "Principles and Techniques of Electron Microscopy: Biological Applications, Volume 2" (M. A. Hayat, ed.), p. 99. Van NostrandReinhold, Princeton, New Jersey, 1972. 2s j. D. Griffith and G. Christiansen, Annu. Rev. Biophys. Bioeng. 7, 19 (1978). 26 G. W. Seegan, C. A. Smith, and V. N. Schumaker, Proc. Natl. Acad. Sci. U.S.A. 76, 907 (1979).
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gives the best contrast is a 1% aqueous unbuffered solution of uranyl acetate. The samples can also be contrasted by shadowing with platinum. 25 This is the method that we prefer because of the fine grain size of the evaporated platinum and the relatively high degree of contrast that can be obtained. Following adsorption of the sample, the grid is washed and dehydrated by briefly dipping the grid in each of a series of solutions: 10 mM Tris-HCl, pH 7.5, followed by 10, 50, 80, and 100% ethanol solutions. The grids are kept under ethanol until they are ready to be shadowed. For shadowing, they are quickly placed on a rotary table in the shadowing chamber, and without allowing the ethanol to dry (this would result in rehydration of the complexes) the chamber is evacuated. The samples are then rotary shadowed with platinum at an angle of 10°. The platinum is evaporated by passing a current through a tungsten wire wrapped with 1.5 cm of 0.2-mm diameter platinum wire. By shadowing the samples from a single direction rather than rotary shadowing, it is possible to estimate the height of the complexes by examining the length of the shadow that is cast. 27In calculating the height of the particle from the length of the shadow by trigonometry, it is necessary to know the angle of the shadowing. Because the grids are seldom perfectly fiat, internal standards such as gold particles or latex spheres should be used to calculate the angle of the shadow) 8 Although adsorption is much more convenient, samples can also be observed by mounting them on mica and preparing a platinum replica. 28 This procedure usually results in somewhat better preservation of the structure of the complex and, because the mica is fiat, somewhat better definition. The complexes to be observed are mixed with glycerol to give a final glycerol concentration of 20--30% (v/v). The presence of the glycerol reduces drying artifacts. The samples should contain about 0.5 mg/ml protein, but several 2-fold serial dilutions should be made to determine the optimal concentration. The sample is then sprayed onto freshly cleaved mica cut into pieces that are about 7 x 15 mm. For spraying, we use a Paasche air brush modified by replacing the nozzle with a plastic micropipet tip. About 10/xl of sample is sprayed onto the mica, which is placed 10-20 cm away. Several different samples can be sprayed in succession on separate mica sheets and shadowed together. For shadowing, the samples are dried for 1-2 hr under a vacuum of 10-6 Torr. After drying, the samples are contrasted with platinum as described above. This is followed by evaporating a layer of carbon to provide support. The
27 p. R. Smith and I. E. Ivanov, J. Ultrastruct. Res. 71, 25 (1980). 2s j. M. Tyler and D. Branton, J. Ultrastruct. Res. 71, 95 (1980).
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replicas are then floated off of the mica onto the surface of distilled water, and picked up with a 400-mesh copper grid. When the samples are observed by electron microscopy, changes in the size and shape of the complexes as the lengths of the nucleic acids increase can be seen. These changes can be related to a stepwise buildup of the complex, reflecting the way in which it is assembled. Therefore, an understanding of the structure of the complexes formed should emerge from an analysis of these data. Further information about the structure of the particles can be acquired by observing complexes formed at subsaturating ratios of protein to nucleic acid and complexes formed under different ionic conditions. Oligomer Binding. A considerable amount of information about how hnRNP proteins bind to nucleic acids can be obtained by examining the binding constants as a function of oligonucleotide size for oligonucleotides ranging in size from monomers to about 50-mers. 29 From these data, one can determine the binding site size, the intrinsic association constant, the amount of cooperativity, and the size of the cooperative unit. The relative affinities for different nucleotides and the effect of ionic strength on the binding can also be determined. The protein of interest is combined with a mixture of 5'-end-labeled oligonucleotides obtained by alkaline hydrolysis of a homopolymer (see above). The protein-bound oligonucleotides are separated from unbound ones by passing the mixture through a 3-mm nitrocellulose filter (which can be cut from a sheet with a small hole punch) and washed briefly with the sample buffer. The amount of each oligonucleotide that is bound depends on the protein concentration and the binding constant for the protein and the particular oligonucleotide. The nucleotides bound to the filter are eluted by boiling the filter in a small amount (10/.d) of 1% SDS, and the eluted oligonucleotides are resolved by electrophoresis through a 20% polyacrylamide gel. The oligonucleotides are detected by autoradiography, and their amounts are quantitated by densitometry. To generate titration curves, the binding experiments are done at several protein concentrations, with the oligomers from each experiment being separated on adjacent lanes of the gel. The protein concentrations used should border the intrinsic binding constant, and the total molar nucleotide concentrations must be less than the molar protein concentration. The resulting data can be combined into a series of titration curves, one for each nucleotide that can be resolved by the gel. By analyzing these titration curves, values for the binding site size and intrinsic binding constant can be calculated. The magnitude of protein-protein interactions 29 j. T. SchOneich and J. O. Thomas, in preparation.
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(cooperativity) can also be evaluated, since these interactions will result in an increase in the affinity for oligomers that are long enough to accommodate two or more proteins. 6 Since the strength of ionic interactions between proteins and nucleic acids is dependent on the salt concentration, the ionic character of the binding can be assessed by determining the intrinsic binding constant (and degree of cooperativity) as a function of ionic strength. 3°
Other Physical Methods In this chapter, we have discussed several techniques for examining the interactions between hnRNP proteins and nucleic acids, and the structures of the complexes that they form. We have emphasized an approach in which the structures are examined as a function of the size of the nucleic acid in the complex. In addition, some other physical techniques have been widely used, and will probably continue to play an important role. Nitrocellulose binding assays are useful for determining nucleotide preferences of hnRNP proteins. 11-14,31 UV absorption spectroscopy and circular dichroism are employed for examining the helix-destabilizing properties of proteins, H-14 and fluorescence quenching is of value for measuring the thermodynamic parameters of the interactions.17 3o M. T. Record, T. M. Lohman, and P. de Haseth, J. Mol. Biol. 107, 145 (1976). ~l A. B. Sachs and R. D. K o m b e r g , Mol. Cell. Biol. 5, 1993 (1985).
[26] I m m u n o l o g i c a l M e t h o d s for P u r i f i c a t i o n a n d Characterization of Heterogeneous Nuclear Ribonucleoprotein Particles
By SERAFIN PllqOL-ROMA, YANG DO CHOI, and GIDEON DREYFUSS Heterogeneous nuclear RNAs (hnRNAs) are associated in the cell with specific proteins to form hnRNA-ribonucleoprotein (hnRNP) complexes, also referred to as hnRNP particles (reviewed by Dreyfussl). hnRNP particles are one of the major components of the nucleus. The proteins of the hnRNP particles are as abundant as his'tones in the nucleus of growing cells, and they comprise approximately 80% of the mass of hnRNP particles, hnRNP complexes can be isolated from vertebrate nu1 G. Dreyfuss, Annu. Rev. Cell Biol. 2, 459 (1986).
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a. For the in vitro assembly of 40 S monoparticles, the added RNA or single-stranded DNA should be between 685 and 726 nt in length or integral multiples of 700 nt for the assembly of dimers and oligomeric complexes. Other RNA lengths will support the assembly of nonstoichiometric complexes which sediment through the gradients. The core proteins of 40 S hnRNP do not assemble in a sequence-dependent manner in vitro, and nucleotide triphosphates are not required as an energy source. b. For correct particle reassembly, the exogenous RNA should be added at 0.7 times the measured OD of the pooled and dialyzed sample. In other words, add 0 . 7 0 D units (28/xg) of exogenous RNA per OD unit of isolated hnRNP. Correct particle assembly will occur over the range 10-20 : I for protein to RNA. Reactant concentration has no significant affect on particle assembly.
[25] P h y s i c a l M e t h o d s for C h a r a c t e r i z a t i o n o f H e t e r o g e n e o u s Nuclear Ribonucleoprotein Complexes
By JERZY T. SCHONEICH a n d JOHN O. THOMAS The major components of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are transcripts of RNA polymerase II, a rather complex set of proteins, and small nuclear (sn) RNAs2 ,2 The sizes of the complexes and their shapes as judged from electron micrographs depend to a large degree on the method used for their preparation. The largest complexes observed sediment at about 200 S. Complexes of this size can be obtained by sonicating nuclei in the presence of RNase inhibitors. 3 In the presence of an RNase activity, either endogenous or an added nuclease, these large complexes are degraded, yielding smaller complexes that sediment in a rather broad band at about 40 S. These complexes contain RNA fragments that range in size from about I00 to about 1000 nucleotides (nt). 4 When viewed by electron microscopy, these particles have an S. Y. Chung and J. Wooley, Proteins: Struct. Funct. Genet. 1, 195 (1986). 2 G. Dreyfuss, Annu. Reo. Cell Biol. 2, 459 (1986). a R. Sperling, P. Spann, D. Often, and J. Sperling, Proc. Natl. Acad. Sci. U.S.A. 83, 6721 (1986). 4 y. D. Choi and G. Dreyfuss, Proc. Natl. Acad. Sci. U.S.A. 81, 7471 (1984).
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average diameter of about 20--25 nm, although they are rather heterogeneous in size and shape. 5 Because of the heterogeneous nature of hnRNP complexes, it is difficult to apply directly the physical methods traditionally used for elucidating the structures of macromolecules. One alternative is to study complexes reconstituted with nucleic acids of defined sizes and to examine the properties of the reconstituted complexes as a function of the size of nucleic acid that is in the complex. Several approaches for doing this are described here.
Principles o f Analysis The principle of this approach is to examine the structures formed between proteins and nucleic acids of defined lengths. Changes in the size and shape of the complexes as the lengths of the nucleic acids increase can be related to a stepwise buildup of the complexes and will reflect their structure and the way in which they are assembled. Depending on the size of nucleic acid that is chosen and the method that is used for analysis, several questions can be addressed. By studying the binding to a series of small oligonucleotides of increasing sizes, one can determine binding site sizes and binding constants and also measure cooperative interactions between neighboring proteins. 6 Also, by examining the effects of solvents on these parameters, some information about the nature of the binding forces (such as the number of ionic interactions) that are involved can be obtained. By using longer nucleic acids as substrates, higher order structures that involve several or many proteins can be examined. In particular, the size and composition of cooperative units can be determined by sedimentation and electrophoresis, and the molecular organization of the complexes can be observed by electron microscopy.
Preparation o f Defined-Length Nucleic Acids RNAs of defined lengths and sequences can be easily obtained by in vitro transcription. A number of procedures using various promoters have been described, 7,8 and several kits are commercially available. For some analyses a heterogeneous mixture of RNAs of various sizes is desirable. This can be produced either by limited alkaline hydrolysis of a transcript (see below) or by synthesizing the RNA in the presence of a limiting 5j. Wooley,s. Y. Chung,J. Wall, and W. LeStourgeon,Biophys. J. 49, 17 (1986). 6j. D. McGheeand P. H. Von Hippel, J. Mol. Biol. 86, 469 (1974). 7 D. A. Melton,P. A. Krieg,M. R. Rebagliati,T. Maniatis,K. Zinn,and M. Green,Nucleic Acids Res. 12, 7035 (1984). 8 E. T, Schenborn and R. C. Mierendorf, Jr., Nucleic Acids Res. 13, 6223 (1985).
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amount of nucleotide, which results in premature terminations and a distribution of RNA sizes. Although the hnRNP proteins are found in association with hnRNA, they also appear to be capable of binding single-stranded DNA. 9-14Large quantities of defined sizes of single-stranded DNAs can be obtained by denaturing DNA fragments generated by restriction enzymes. These can be end-labeled by the Klenow fragment of DNA polymerase 15 prior to denaturation, or they can be labeled with T4 polynucleotide kinase after removal of the 5'-phosphate by phosphatase. 15The DNA can be rendered single stranded by dissolving it in 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, heating it in a boiling water bath, and quickly cooling it on ice. Homopolymers are useful for reconstitution because they provide simplified model systems that are homogeneous in composition and secondary structure. Homopolymers, which are typically several hundred nucleotides long, can be obtained from a number of commercial sources. Commercial poly(A) is usually longer than poly(U) or poly(C). Shorter homopolymers can be prepared by limited alkaline hydrolysis 16 (for deoxyhomopolymers, a limited digestion with micrococcal nuclease can be used), which leaves a free 5'-hydroxyl that can be conveniently labeled using T4 polynucleotide kinase. To obtain defined sizes of homopolymers, a partially hydrolyzed mixture can be separated by electrophoresing through a polyacrylamide gel, cutting the gel, and eluting the fragments of the desired sizes. To elute the fragments, the end of a plastic micropipet tip is plugged with a small amount of siliconized glass wool, and the end is sealed. The gel piece is placed in the pipet tip and crushed with a glass rod. After crushing, 0.5 ml of 0.5 M ammonium acetate, 1 mM EDTA is added, and the RNA is allowed to elute overnight. The end of the tip is then cut, the solution is drained, and the RNA is precipitated with 2.5 volumes of ethanol. For larger fragments, electroelution also works well. To produce a mixture of RNA fragments by alkaline hydrolysis,16 the RNA or homopolymer in 50 mM sodium carbonate buffer, pH 9.0, is 9 j. M. Pullman and T. E. Martin, J. Cell Biol. 97, 99 (1983). ~0H.-E. Wilk, G. Angeli, and K. P. Sch~fer, Biochemistry 22, 4592 (1983). i1 L. Nowak, D. K. Marvil, J. O. Thomas, M. Boublik, and W. Szer, J. Biol. Chem. 255, 6473 (1980). lz Raziuddin, J. O. Thomas, and W. Szer, Nucleic Acids Res. 10, 7777 (1982). 13 A. Kumar, K. R. Williams, and W. Szer, J. Biol. Chem. 261, 11266 (1986). 14A. Kumar, H. Sierakowska, and W. Szer, J. Biol. Chem. 262, 17126 (1987). 15 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 16 H. Donis-Keller, A. M. Maxam, and W. Gilbert, Nucleic Acids Res. 4, 2527 (1977).
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heated to 65 ° in a microcentrifuge tube. After 3, 10, and 25 min, samples are placed on ice and neutralized with 1/10 volume of 2.5 M sodium acetate, 10 mM MgCIE, pH 5.2. Because the rate of degradation is very sensitive to the exact conditions used, particularly the pH, it is necessary to check the extent of hydrolysis of each sample. This is done by 5'-endlabeling a small portion and analyzing it by polyacrylamide gel electrophoresis and autoradiography. Reconstitution with Total h n R N P Proteins
Methods for forming reconstituted hnRNP complexes using exogenously added nucleic acids have been described. 9a° All of these methods suffer from the lack of a suitable structural or functional assay for "native" hnRNP complexes, so it is never certain how faithfully the reconstituted particles reflect the structure of the complexes that are formed in vivo. Nonetheless, in vitro reconstitution studies do provide a handle for investigating the RNA-protein and protein-protein interactions that can occur during the formation of the structures. This is a useful and perhaps essential framework for examining these rather multifarious particles. The simplest method for reconstitution is to digest the endogenous RNA and then add the desired nucleic acid in its place. The proteins associate spontaneously with the added nucleic acid. The hnRNP complexes [1 mg/ml protein in l0 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1), 50 mM NaC1, 1 mM CaC12, 1 mM dithiothreitol (pH 7.6)] are digested with 2.5 units/ml micrococcal nuclease. After 10 rain at 37°, the nuclease is inactivated by adding EDTA to 5 mM. The exogenous RNA is then added to give a protein to nucleotide molar ratio between I : 6 and 1 : 12. The composition of the buffer is not particularly critical, although elevated salt concentrations tend to favor the dissociation of most nucleic acid-protein complexes and very low concentrations of salt may lead to the precipitation of hnRNP proteins. Magnesium does not appear to have much influence on the formation of the complexes. Dithiothreitol (DTT) is routinely included since the free proteins tend to precipitate in its absence. 13,14 For the reconstitution of complexes with poly(A), the endogenous RNA can be digested with RNase A. Since poly(A) is not degraded by RNase A, the complete inhibition of the enzyme is not a consideration as it is with micrococcal nuclease. Reconstitution with Individual Proteins
A few hnRNP proteins have been purified and partially characterized. n-14,17 They appear to share the ability to bind to RNA (and single17 F. Cobianchi, R. L. Karpel, K. R. Williams, V. Notario, and S. H. Wilson, J. Biol. Chem. 263, 1063 (1988).
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stranded DNA) and form condensed nucleic acid-protein complexes. 12,1a,19 The best characterized complexes are those formed with HD40, the major hnRNP protein of Artemia salina. HD40 condenses nucleic acids into helices that are only marginally stable; on average, the helix formed by the nucleoprotein complex is only about 2] turns. 18 The structure of the hnRNP complexes, and the way in which the nucleic acids are condensed, can be examined by observing the particles formed when the proteins associate with nucleic acids of increasing lengths. The effect of varying the protein to nucleotide ratio and the salt conditions used for reconstitution can also be examined to give additional information about the structure of the complexes and the forces involved in their formation. Homopolymers of defined sizes are good substrates for these studies since complications arising from sequence specificities of the proteins and differences in secondary structures of the nucleic acids can be minimized. Although the complexes can be observed with almost any physical technique, electron microscopy, electrophoresis, and hydrodynamic methods are particularly informative. Typically, the nucleic acid-protein complexes are formed simply by mixing the protein and nucleic acid. It is important to keep the protein concentration as high as possible to assure that the complex is fully saturated (the complexes that have been examined have intrinsic association constants of about 105-106 M - l ) . llA7 Following the formation of the complex, it can be analyzed as described below.
Analysis of Reconstituted Complexes Sedimentation. As mentioned above, sedimentation has been widely used for the isolation and characterization of hnRNP complexes. Sedimentation can also be used in conjunction with electrophoresis for the analysis of hnRNP complexes reconstituted with either defined-length RNAs or with a heterogeneous mixture of RNAs of various sizes. Following reconstitution (see above), the mixture is layered on top of a 10-30% (w/w) sucrose gradient made with a buffer containing 10 mM Tris-HC1, 25 mM NaC1, 5 mM EDTA, 1 mM DTT, pH 7.6, and centrifuged. For sucrose gradients made on a percent (w/w) basis, a good approximation of the sedimentation coefficient as a function of the position in the gradient can be easily calculated, 2° although a more exact determination should be made by comparing the rate of sedimentation with ribosomal markers. For analytical purposes, centrifugation in a Beckman SW 50.1 rotor is ~8j. O. Thomas, S. K. Glowacka, and W. Szer, J. Mol. Biol. 171, 439 (1983). ~9j. T. Sch6neich and J. O. Thomas, unpublished observations (1988). 2o C. R. McEwen Anal. Biochem. 20, 114 (1967).
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convenient. After a 100-min centrifugation at 5° at 49,000 rpm, 60 S material should be located near the middle of the gradient. Following fractionation, the gradients can be analyzed by several methods. The simplest is to plot the distribution of radioactivity through the gradient. More information can be obtained, particularly if heterogeneous RNA is used, if the RNA is isolated from each fraction by phenol extraction and ethanol precipitation and then analyzed by electrophoresis. Separation of the RNA on 3.5% polyacrylamide-8 M urea gels is convenient for resolving RNAs between 0. l and 2 kilobases (kb) in size. For smaller RNAs, the concentration of acrylamide can be increased up to 20%. For larger RNAs, agarose-formaldehyde gels zl should be used. After separating the RNAs on gels, the size distribution of the RNAs in each fraction can be quantitated by densitometry of autoradiograms. From one experiment in which a broad size range of RNAs is used, the sedimentation behavior of complexes incorporating this population of RNAs can be determined. This enables a direct comparison to be made between complexes formed with RNAs of many different sizes under the exact same conditions. In addition to an analysis of the RNAs in each fraction of the gradient, the protein composition of each fraction can be determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. 22 For detection, the proteins can be stained either with Coomassie brilliant blue or w i t h silver. 23 The silver stain is much more sensitive, but it should be noted that with silver the major hnRNP proteins are less intensely stained relative to the other protein components of the hnRNP complexes than they are with Coomassie brilliant blue. A serious problem with examining the proteins present in complexes separated by sedimentation through sucrose gradients is that the hnRNP proteins readily aggregate in the absence of nucleic acid, particularly in low-salt buffers in the absence of DTT. 13,14This makes it difficult to distinguish between nucleic acid-protein complexes and aggregates of free proteins that might cosediment with the complexes. Analytical ultracentrifugation is useful for examining the interactions between purified hnRNP proteins and nucleic acids. From sedimentation equilibrium studies, the molecular weights of complexes formed with different sizes of nucleic acids can be determined, and from the change in molecular weight as a function of nucleic acid size, the excluded binding site size can be calculated.~8 The molecular weights can also be used in conjunction with sedimentation coefficients, determined by sedimenta21 H. Lehrach, D. Diamond, J. M. Wozney, and H. Boedtker, Biochemistry 16, 4743 (1977). 2~ U. K. Laemmli, Nature (London) 227, 680 (1970). 23 C. R. Merril, D. Goldman, S, A. Sedman, and M. H. Ebert, Science 211, 1437 (1981).
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tion velocity experiments using the same samples, to estimate frictional coefficients and axial ratios of the complexes.la Electrophoresis on Nondenaturing Gels. Electrophoresis through nondenaturing agarose gels provides an alternative to sedimentation for the separation of reconstituted hnRNP complexes. A major advantage of electrophoresis over sedimentation is that there is a clear separation of the RNA-protein complexes from free proteins and from unbound RNAs. This enables one to characterize both the RNA and protein components of the complexes that are formed. Reconstituted complexes [in 20 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (HEPES), 50 mM NaC1, 5% (v/v) glycerol, 1 mM DTT, 0.001% bromphenol blue (pH 7.6)] are loaded onto a 1% agarose gel [in 20 mM HEPES, 25 mM NaCl, 5% (v/v) glycerol, 1 mM DTT (pH 7.6)]. Electrophoresis is conducted in the cold at 3.5 V/cm until the dye front has migrated about 80% of the length of the gel. The running buffer [50 mM Tris-HC1, 25 mM NaC1, 1 mM DTT (pH 7.6)] should be continuously recirculated. In nondenaturing gels, the free hnRNP proteins remain near the origin. Free nucleic acids and nucleic acid-protein complexes move toward the positive electrode, with the free nucleic acids migrating much faster than nucleic acid-protein complexes. 19Separation of the complexes is based on a combination of size, shape, and net charge. The distribution of the RNA and protein components throughout the nondenaturing gel can be analyzed on second dimension gels: a polyacrylamide gel containing 8 M urea for the analysis of RNA or an SDS-polyacrylamide gel for the analysis of proteins. A disadvantage of using electrophoresis for the resolution of the complexes is the fact that one cannot a priori determine the relationship between sedimentation coefficients and mobilities on gels. Rather, it is necessary to correlate empirically the electrophoretic mobilities of the complexes with their sedimentation coefficients. Electron Microscopy. Electron microscopy is potentially an extremely powerful tool for examining the structure of macromolecular complexes. However, since the hnRNP complexes are rather heterogeneous, the value of electron microscopy has been limited. The problem of sample heterogeneity can be reduced by examining complexes reconstituted with defined sizes of oligonucleotides. This approach can be particularly powerful if the morphology of the complex is examined as a function of the size of the nucleic acid used for reconstitution. For electron microscopy, complexes are reconstituted with the desired nucleic acid as described above. Complexes containing nucleic acids longer than 100 nt can be easily visualized, but structures containing fewer than 50 nt are difficult to distinguish reliably from background
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debris. Complexes reconstituted from crude hnRNP proteins are also difficult to analyze because of a large background of proteins and protein aggregates. In order to preserve the structure of the complexes during the subsequent processing steps, it may be desirable to fix them by adding 1/20 volume of 8% glutaraldehyde to the reconstituted complexes.~8 On the other hand, the presence of the fixative might alter the structure of the complexes and may also lead to the formation of protein aggregates if fixation is excessive. Because of these problems, it is best to examine and compare both fixed and unfixed complexes. In our experience, there is usually little difference. Specimens can be mounted either by spraying them onto a mica sheet or carbon film or by direct adsorption onto a carbon film. Adsorption is much easier. For adsorption, thin carbon films on 400-mesh copper electron microscope grids 24 are used. The carbon-coated grids are generally hydrophobic and do not interact with the nucleic acid-protein complexes. There are several methods for making the grids hydrophilic. The easiest and most satisfactory method for nucleic acids coated with proteins is to activate the grids by placing them in a glow discharge. 25 We generate a glow discharge by passing a 55 mA current through a 80-120 mTorr vacuum. The grids should be kept in the discharge for about 8 min. Insufficient activation will not render the grids hydrophilic, and overactivation will result in increased background contamination. Consequently, it is best to determine empirically the optimal conditions for each particular apparatus. The activated grids should be used the same day. The nucleoprotein complexes are mounted on the activated grids simply by placing a 5- to 10-/xl drop of sample on the grids and allowing it to adsorb for about 30 sec. The amount of sample that is adsorbed onto the grid is dependent on the adsorption time, so dilute samples may require longer times. For negative staining, the droplet is washed off of the grid with several drops of 10 mM Tris-HCl, pH 7.5, applied rapidly with a Pasteur pipet. This is followed by several drops of stain. The stain is then immediately removed by touching a piece of paper towel or filter paper to the edge of the grid. When done properly, a thin film of stain will remain on the grid. This will quickly dry in the air, and the grids can be observed immediately. A number of stains are commonly used for the negative contrasting of macromolecules. 24,26 In our experience, the stain which 24 R. H. Haschemeyer and R. J. Myers, in "Principles and Techniques of Electron Microscopy: Biological Applications, Volume 2" (M. A. Hayat, ed.), p. 99. Van NostrandReinhold, Princeton, New Jersey, 1972. 2s j. D. Griffith and G. Christiansen, Annu. Rev. Biophys. Bioeng. 7, 19 (1978). 26 G. W. Seegan, C. A. Smith, and V. N. Schumaker, Proc. Natl. Acad. Sci. U.S.A. 76, 907 (1979).
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gives the best contrast is a 1% aqueous unbuffered solution of uranyl acetate. The samples can also be contrasted by shadowing with platinum. 25 This is the method that we prefer because of the fine grain size of the evaporated platinum and the relatively high degree of contrast that can be obtained. Following adsorption of the sample, the grid is washed and dehydrated by briefly dipping the grid in each of a series of solutions: 10 mM Tris-HCl, pH 7.5, followed by 10, 50, 80, and 100% ethanol solutions. The grids are kept under ethanol until they are ready to be shadowed. For shadowing, they are quickly placed on a rotary table in the shadowing chamber, and without allowing the ethanol to dry (this would result in rehydration of the complexes) the chamber is evacuated. The samples are then rotary shadowed with platinum at an angle of 10°. The platinum is evaporated by passing a current through a tungsten wire wrapped with 1.5 cm of 0.2-mm diameter platinum wire. By shadowing the samples from a single direction rather than rotary shadowing, it is possible to estimate the height of the complexes by examining the length of the shadow that is cast. 27In calculating the height of the particle from the length of the shadow by trigonometry, it is necessary to know the angle of the shadowing. Because the grids are seldom perfectly fiat, internal standards such as gold particles or latex spheres should be used to calculate the angle of the shadow) 8 Although adsorption is much more convenient, samples can also be observed by mounting them on mica and preparing a platinum replica. 28 This procedure usually results in somewhat better preservation of the structure of the complex and, because the mica is fiat, somewhat better definition. The complexes to be observed are mixed with glycerol to give a final glycerol concentration of 20--30% (v/v). The presence of the glycerol reduces drying artifacts. The samples should contain about 0.5 mg/ml protein, but several 2-fold serial dilutions should be made to determine the optimal concentration. The sample is then sprayed onto freshly cleaved mica cut into pieces that are about 7 x 15 mm. For spraying, we use a Paasche air brush modified by replacing the nozzle with a plastic micropipet tip. About 10/xl of sample is sprayed onto the mica, which is placed 10-20 cm away. Several different samples can be sprayed in succession on separate mica sheets and shadowed together. For shadowing, the samples are dried for 1-2 hr under a vacuum of 10-6 Torr. After drying, the samples are contrasted with platinum as described above. This is followed by evaporating a layer of carbon to provide support. The
27 p. R. Smith and I. E. Ivanov, J. Ultrastruct. Res. 71, 25 (1980). 2s j. M. Tyler and D. Branton, J. Ultrastruct. Res. 71, 95 (1980).
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replicas are then floated off of the mica onto the surface of distilled water, and picked up with a 400-mesh copper grid. When the samples are observed by electron microscopy, changes in the size and shape of the complexes as the lengths of the nucleic acids increase can be seen. These changes can be related to a stepwise buildup of the complex, reflecting the way in which it is assembled. Therefore, an understanding of the structure of the complexes formed should emerge from an analysis of these data. Further information about the structure of the particles can be acquired by observing complexes formed at subsaturating ratios of protein to nucleic acid and complexes formed under different ionic conditions. Oligomer Binding. A considerable amount of information about how hnRNP proteins bind to nucleic acids can be obtained by examining the binding constants as a function of oligonucleotide size for oligonucleotides ranging in size from monomers to about 50-mers. 29 From these data, one can determine the binding site size, the intrinsic association constant, the amount of cooperativity, and the size of the cooperative unit. The relative affinities for different nucleotides and the effect of ionic strength on the binding can also be determined. The protein of interest is combined with a mixture of 5'-end-labeled oligonucleotides obtained by alkaline hydrolysis of a homopolymer (see above). The protein-bound oligonucleotides are separated from unbound ones by passing the mixture through a 3-mm nitrocellulose filter (which can be cut from a sheet with a small hole punch) and washed briefly with the sample buffer. The amount of each oligonucleotide that is bound depends on the protein concentration and the binding constant for the protein and the particular oligonucleotide. The nucleotides bound to the filter are eluted by boiling the filter in a small amount (10/.d) of 1% SDS, and the eluted oligonucleotides are resolved by electrophoresis through a 20% polyacrylamide gel. The oligonucleotides are detected by autoradiography, and their amounts are quantitated by densitometry. To generate titration curves, the binding experiments are done at several protein concentrations, with the oligomers from each experiment being separated on adjacent lanes of the gel. The protein concentrations used should border the intrinsic binding constant, and the total molar nucleotide concentrations must be less than the molar protein concentration. The resulting data can be combined into a series of titration curves, one for each nucleotide that can be resolved by the gel. By analyzing these titration curves, values for the binding site size and intrinsic binding constant can be calculated. The magnitude of protein-protein interactions 29 j. T. SchOneich and J. O. Thomas, in preparation.
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(cooperativity) can also be evaluated, since these interactions will result in an increase in the affinity for oligomers that are long enough to accommodate two or more proteins. 6 Since the strength of ionic interactions between proteins and nucleic acids is dependent on the salt concentration, the ionic character of the binding can be assessed by determining the intrinsic binding constant (and degree of cooperativity) as a function of ionic strength. 3°
Other Physical Methods In this chapter, we have discussed several techniques for examining the interactions between hnRNP proteins and nucleic acids, and the structures of the complexes that they form. We have emphasized an approach in which the structures are examined as a function of the size of the nucleic acid in the complex. In addition, some other physical techniques have been widely used, and will probably continue to play an important role. Nitrocellulose binding assays are useful for determining nucleotide preferences of hnRNP proteins. 11-14,31 UV absorption spectroscopy and circular dichroism are employed for examining the helix-destabilizing properties of proteins, H-14 and fluorescence quenching is of value for measuring the thermodynamic parameters of the interactions.17 3o M. T. Record, T. M. Lohman, and P. de Haseth, J. Mol. Biol. 107, 145 (1976). ~l A. B. Sachs and R. D. K o m b e r g , Mol. Cell. Biol. 5, 1993 (1985).
[26] I m m u n o l o g i c a l M e t h o d s for P u r i f i c a t i o n a n d Characterization of Heterogeneous Nuclear Ribonucleoprotein Particles
By SERAFIN PllqOL-ROMA, YANG DO CHOI, and GIDEON DREYFUSS Heterogeneous nuclear RNAs (hnRNAs) are associated in the cell with specific proteins to form hnRNA-ribonucleoprotein (hnRNP) complexes, also referred to as hnRNP particles (reviewed by Dreyfussl). hnRNP particles are one of the major components of the nucleus. The proteins of the hnRNP particles are as abundant as his'tones in the nucleus of growing cells, and they comprise approximately 80% of the mass of hnRNP particles, hnRNP complexes can be isolated from vertebrate nu1 G. Dreyfuss, Annu. Rev. Cell Biol. 2, 459 (1986).
METHODS IN ENZYMOLOGY,VOL. 181
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