[ 2 7 ] PHOTOAFFINITY CROSS-LINKING OF R N A - P R O T E I N 383

[27] Photoaf f in i ty Cross-Linking M e t h o d s for S tudy ing R N A - P r o t e i n Interact ions

By M I C H E L L E M . H A N N A

Photochemical cross-linking can be used to characterize RNA-protein interactions in ribonucleoprotein (RNP) complexes, which exist tran- siently during transcription, translation, splicing, or RNA processing or as stable structural and catalytic components of cells. Experiments can be designed to answer questions as simple as whether a RNA-protein inter- action exists, or as complex as which nucleotide(s) or amino acid(s) are involved in a known RNP complex. Photochemical cross-linking "traps" weak or transient RNA-protein associations, which might not survive isolation procedures, such as immunoprecipitation, gel filtration, or filter binding, permitting detailed structural studies of these complexes.

There are a number of approaches that can be taken in photochemical crosslinking, and the choice of an approach depends on the specific ques- tion to be answered, the purity of the proteins being studied, the sequence and structure of the RNA, and the strength of the interaction between the RNA and protein. One method, which has been frequently used, involves the direct irradiation of a RNP complex with short wavelength ultraviolet (UV) light. This method relies on the direct excitation of nucleotides or amino acids, depending on which wavelength is used, to generate chemi- cally reactive species. While this nonspecific labeling may show whether a protein-nucleic acid interaction exists, there can be problems with this method, the most serious being the degradation of some proteins by the irradiation itself.

An alternate approach involves the use of photosensitive cross-linking reagents. These reagents contain groups that are chemically inert in the absence of light, but can be easily converted to chemically reactive spe- cies by irradiation, sometimes at long wavelengths in the UV and visible spectrums, which will not nick protein or nucleic acid. Even when short- wavelength UV light must be used, the high reactivity of these groups can result in cross-linking, before considerable damage is done to the protein or nucleic acids.

Copyright © 1989 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 180 All rights of reproduction in any form reserved.

384 RNA INTERACTIONS [27]

Approaches to P r o t e i n - R N A Cross-Linking

Addition of Bifunctional Cross-Linking Reagent to Ribonucleoprotein Complex Containing Unmodified RNA and Protein

Bifunctional cross-linking reagents contain two cross-linking groups separated by linkers of about 5 -20 A. Some linkers contain bonds that can be chemically cleaved by thiol or periodate. Both cross-linking groups may be photoreactive, or more commonly, these reagents contain one chemically reactive group and one photoreactive group. Most groups con- tain photoreactive aryl azides as the cross-linking functional group. ~ The half-life of the photolytically produced reactive species, the nitrene, is on the order of a millisecond, and its insertion reactions are highly nonspe- cific. 2 This nonspecificity is particularly useful, when probing the environ- ment of a protein or nucleic acid, as there need not be a specific functional group within contact of the nitrene to achieve cross-linking. The wave- length required for photolysis of unsubstituted aryl azides is between 260 and 290 nm; however, substitutions can shift the excitation maxima to longer wavelengths. Addition of a nitro group to most aryl azides shifts the excitation maxima to between 300 and 460 nm. These substituted azides can undergo cross-linking at wavelengths that will not damage proteins or nucleic acids. Several of these bifunctional cross-linking agents are listed in Table 1. 3-30

G. W. J. Fleet, R. R. Porter, and J. R. Knowles, Nature (London) 224, 511 (1969). 2 A. K. Schrock and G. B. Schuster, J. Am. Chem. Soc. 106, 5228 (1984). a p. A. S. Smith and B. B. Brown, J. Am. Chem. Soc. 73, 2438 (1951). 4 A. Reiser, H. M. Wagner, R. Morley, and A. Bowes, Trans. Faraday Soc. 63, 2403 (1967). 5 R. B. Mikkelsen and D. F. H. Wallach, J. Biol. Chem. 251, 7413 (1976). 6 W. L. Denfler, M. M. Pratt, and R. E. Stephens, J. Cell Biol. 84, 381 (1980). 7 R. V. Lewis, M. F. Roberts, E. A. Dennis, and W. S. Allison, Biochemistry 16, 5650

(1977). s y . A. Vladimirov, D. J. Roshchupkin, and E. E. Fesenko, Photochem. Photobiol. 11, 227

(1970). 9 D. J. Kiehm and T. H. Ji, J. Biol. Chem. 252, 8524 (1977). l0 M. Haeupfle, M. L. Aubert, J. Djiane, and J.-P. Kraehenbuhl, J. Biol. Chem. 258, 305

(1983). H A. M. Tometsko and J. Turula, Int. 7. Pep. Protein Res. 8, 331 (1976). 12 B. D. Burleigh, W.-K. Liu, and D. N. Ward, 7. Biol. Chem. 253, 7179 (1978). 13 R. E. Galardy, L. C. Craig, J. D. Jamieson, and M. P. Printz, 7. Biol. Chem. 249, 3510

(1974). t4 C. W. T. Yeung, M. L. Mottle, and C. C. Yip, Biochemistry 19, 2196 (1980). 15 T. H. Ji, J. Biol. Chem. 252, 1566 (1977). 16 K. Sutoh and F. Matsuzaki, Biochemistry 19, 3878 (1980). 17 T. H. Ji and I. Ji, Anal. Biochem. 121, 286 (1982). is I. Ji, J. Shin, and T. H. Ji, Anal. Biochem. 151, 348 (1985).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 385

These reagents can be used to cross-link molecules that have not been modified with reactive groups and therefore exist in their native confor- mations, provided care has been taken in their isolation. Cross-linking reactions are prepared by combining unmodified protein and RNA (either as purified components or in cellular fractions), using conditions that are optimal for association of the RNA and protein. This allows the RNP complex to form in the absence of added chemical reagents not normally found in the system. The advantage of this method is that unmodified macromolecules are more likely to form complexes more similar to in vivo complexes than modified RNA or protein. After formation of the RNP complex, the bifunctional cross-linking reagent is added to the reaction (in dim light), and covalent linkages are formed between the RNA and pro- tein, by adjusting reaction conditions for the chemical cross-linker and/or by excitation of the photoreactive group(s) with light. Cross-linker can be added directly as solid to the RNA-protein solution, if sufficiently solu- ble, or can be dissolved in a minimal amount of organic solvent [dimethyl sulfoxide (DMSO) or dimethylformamide (DMF)] and then can be added to the aqueous solution. Most of these bifunctional reagents hydrolyze rapidly in water and should not be stored as aqueous solutions. Most chemical modifications are complete in about 30 min at room temper- ature.

Disadvantages of this approach include the following.

I. Use of cross-linkers containing two photoreactive groups requires a trimolecular reaction to achieve cross-linking. The cross-linking reagent must become positioned as a bridge between the protein and RNA in such a way that one cross-linking group is within a few angstroms of each of these macromolecules. On irradiation, both groups must undergo suc-

t9 V. Chowdhry, R. Vaughan, and F. H. Westheimer, Proc. Natl. Acad. Sci. U.S.A. 73, 1406 (1976).

2o V. Chowhdry and F. H. Westheimer, Annu. Rev. Biochem. 48, 293 (1979). 21 E. F. Vanin and T. H. Ji, Biochemistry 20, 6754 (1981).

J. U. Baenziger and D. Fiete, J. Biol. Chem. 257, 4421 (1982). 23 D. A. Zarling, J. A. Miskimen, D. P. Fan, E. K. Fujimoto, and P. K. Smith, J. lmmunol.

128, 251 (1982). 24 S. M. Jung and M. Moroi, Biochim. Biophys. Acta 761, 152 (1983). 2~ K. Ballmer-Hofer, V. Schlup, P. Burn, and M. M. Burger, Anal. Biochem. 126, 246

(1982). 26 H. Wollenweber and D. C. Morrison, J. Biol. Chem. 260, 15068 (1986). 27 S. H. Hixson and S. S. Hixson, Biochemistry 14, 4251 (1975). 2, M. Erecinska, Biochem. Biophys. Res. Commun. 76, 495 (1977). 29 R. B. Moreland, P. K. Smith, E. K. Fijimoto, and M. E. Dockter, Anal. Biochem. 121,

321 (1982). 3o C. K. Huang and F. M. Richards, J. Biol. Chem. 252, 5514 (1977).

386 RNA INTERACTIONS [27]

TABLE I BIFUNCTIONAL PHOTOCROSS-L1NKING REAGENTS

Reagent, Specificity b Cleavage Advantages c Reference(s)

DABP DAN DTBPA ANB-NOS EADB FNPA HSAB MABI NHS-ASA PNP-DTP SADP SANPAH Sulfo-SADP SAND

Nonspecific Nonspecific Nonspecific Amines Amines Amines Amines Amines Amines Amines Attunes

Thiols

Thiols

Thiols

Thiols Thiols

Ex = 320-350 nm

Ex = 320-350 nm

Iodinatable

3-5 3-5 3-6 7,8 9, 10 11, 12 13, 14 15, 16 17, 18 19, 20 21-24 22, 25 21-23 7

Amines Ex = 320-350 nm Attunes Water soluble Amines Water soluble

Ex = 320-350 nm Sulfo-SANPAH Amines Water soluble 22, 25

Ex = 320-350 nm SASD Amines Tbiols Water soluble 26

Iodinatable APB Thiols 27, 28 APTP Thiols Thiols 9, 29 DNCO Thiols Thiols Ex = visible light 30

° ANB-NOS, N-5-Azido-2-nitrobenzoyloxysuccinimide; APB, p-azidophenacyl bro- mide; APTP, N-(4-azidophenylthio)phthalimide; DABP; 4,4'-diazidobiphenyl; DAN, diazidonaphthalene; DNCO, di-N-(2-nitro-4-azidophenyl)cystamine-S-S-dioxide; DTBPA, 4,4'-dithiobisphenylazide; EADB, ethyl 4-azidophenyl-l,4-dithiobutyrimi- date-HCl; FNPA, 4-fluoro-3-nitrophenylazide; HSAB, N-hydroxysuccinimidyl-4- azidobenzoate; MABI, methyl-4-azidobenzoimidate; NHS-ASA, N-hydroxysuccini- midyl-4-azidosalicylic acid; PNP-DTP, p-nitrophenyl-2-diazo-3,3,3-trifiuoropropio- nate; SADP, N-succinimidyl (4-azidophenyl)-l,Y-dithiopropionate; SAND, sulfosuc- cinimidyl 2-(m-azido-o-nitrobenzamido)ethyl-l,Y-dithiopropionate; SANPAH, N-suc- cinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate; SASD, salfosuccinimidyl 2-(p- azidosalicylamido)ethyl-l,2'-dithiopropionate; sulfo-SANPAH, sulfosuccinimidyl 6- (4'-azido-2'-nitrophenylamino)hexanoate; sulfo-SADP, sulfosuccinimidyl(4-azido- phenyldithio)propionate.

b Specificity refers to the group modified by the chemical cross-linking groups on hetero- bifunctional reagents. Nonspecific means that both cross-linking groups are photoreac- tive and react nonspecifically with many functional groups.

c Ex, Optimal wavelengths for excitation of the photncross-linking groups.

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 387

cessful cross-linking to the macromolecules, as opposed to solvent. As positioning of the cross-linking group is random, intramolecular cross- links within the protein and RNA molecules may be as likely as RNA- protein intermolecular cross-links. Such intramolecular cross-links can severely complicate characterization of cross-linked complexes. These reagents frequently give low cross-linking yields.

2. Use of bifunctional reagents, containing a chemical cross-linking group, can limit the types of molecular interactions that can be identified. These chemical cross-linkers are generally specific for some functional group, typically amines or thiols, and cannot be used to probe regions of molecules that do not contain accessible groups of the necessary type.

3. Chemical cross-linking groups generally require a narrow pH range for optimal reactivity. This is in the basic range (pH 7-11) for amine modifying groups and in the slightly basic range (pH 7-8) for thiol modify- ing groups. As most RNA molecules contain no thiol groups, and many proteins contain few, if any, free-thiol groups, amine modifying reagents may be the only choice for a given system. The pH required for efficient modification may not be that in which the RNP complex is stable or can exist in its native conformation.

4. Since bifunctional reagents cannot be targeted to the protein or RNA molecule of interest, this approach is generally useful only with purified components. Since these reagents can react with almost any mol- ecule the reagents encounter, cross-linking in crude extracts can lead to formation of large aggregates, which complicates identification and isola- tion of the desired complex.

5. Most of the bifunctional cross-linkers are only slightly soluble in water and must be dissolved in organic solvents. Addition of such organic solvents to biological solutions may have some effect on normal RNP complex formation.

6. Buffers containing amines cannot be used with amine-modifying reagents. Tris- or glycine-containing buffers must be changed to acetate-, phosphate-, borate-, or citrate-containing buffers. Thiols, such as dithiothreitol (DTr) or 2-mercaptoethanol, cannot be used in buffers when thiol-modifying reagents or reagents containing disulfide bonds are used. These changes may affect normal RNA-protein interactions.

Placement o f Photoreactive Cross-Linking Group on Purified Protein and Addition o f This Modified Protein to Unmodified RNA

The reagents listed in Table I, which contain both chemical and photo- chemical cross-linking groups, can also be used to modify purified pro- teins or purified nucleic acids. The use of such reagents and their mecha-

388 RNA INTERACTIONS [27]

nisms of cross-linking have been thoroughly discussed by others. 31,32 The major limitation of this approach is that one must have both a purified protein and an assay for the activity of that protein. Photoreactive cross- linking groups can be placed on either thiol or amino groups. Cross-linker- to-protein ratios can be adjusted to add (on the average) only one cross- linking group or several per protein molecule.

Proteins must be dialyzed out of storage buffers, which contain amines and/or thiol, before reaction with the bifunctional cross-linker, as these reagents will react with buffer components as well. Although the cross- linking reagents may require solution conditions, which are not optimal for the protein, in many cases, normal conformations and activities will be regained, when the unreacted reagents are removed by dialysis of the modified protein back into its storage buffer.

The extent of protein modification can be determined by monitoring absorbance of the protein-cross-linker adduct, when a photoreactive group has been chosen that absorbs light above 300 nm. After covalent attachment of the cross-linker to the protein, unreacted reagent is re- moved by dialysis. An aliquot of the modified protein can then be scanned to determine absorbance at the absorption maxima for both the protein and the photoreactive cross-linking group. By knowing the extinction coefficient for both at these wavelengths, one can determine fairly accu- rately the number of cross-linking groups added per molecule. This re- quires a great deal of protein, however. When limited quantities are avail- able, the extent of modification can be estimated by reacting proteins with fluorescent tags specific for the same group to which the photoreactive cross-linker was attached, optimizing conditions for complete protein modification, rather than protein stability. Comparison of the number of fluorescent groups added to cross-linker-modified and unmodified protein provides an estimation of the average number of cross-linking groups per molecule.

Disadvantages of this method include the following.

1. Except in the case in which there may be only one free thiol on a protein, it is impossible to target the cross-linker to one specific amino acid or to ensure that a protein will be modified by only one cross-linking group.

2. Unless large quantities of protein are available, determining the extent of modification can be difficult.

3. Conditions necessary for the modification of the protein may place the protein in a conformation that exposes groups normally buried in the

3t H. Bayley and J. R. Knowles, this series, VoL 46, p. 69. 32 V. Chowdhry and F. H. Westheimer, Annu. Reo. Biochem. 48, 293 (1979).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 389

native structure. Modification of these groups may inhibit the correct refolding of the protein, even after dialysis back into its storage buffer.

4. Removal of thiol (and in some cases, certain amines) may result in irreversible loss of biological activity of the protein, even before exposure to chemical cross-linking reagents.

5. Modification of proteins may lead to loss of activity or loss of the ability to correctly associate with other proteins or assemble into a RNP complex. If chemical modification of the protein has resulted in loss of its normal activity, the contacts the protein makes when added back to RNA may be meaningless. One must assay to determine whether the chemi- cally modified protein still has normal activity or binding properties be- fore proceeding with photocross-linking.

Placement of Photoreactive Group within RNA Molecule and Addition of This Modified RNA to Unmodified Protein

By placing the photoreactive cross-linking group on the RNA mole- cule, it is possible to study RNA-protein interactions with proteins in their native conformations. Care must be taken, however, to maintain the native conformation of the RNA during introduction of the cross-linker. Photoreactive groups can be placed within RNA molecules by two means. The first is similar to method described previously, Placement of Photo- reactive Cross-Linking Group on Purified Protein and Addition of This Modified Protein to Unmodified RNA, and involves the chemical attach- ment of bifunctional reagents to purified RNA molecules. Except in the case of tRNA, where 4-thiouridine can react with thiol-modifying re- agents, the target for modification on RNA would be primary amine groups. Modification of 4-thiouridine with azidophenacyl bromide has been discussed elsewhere, 33,34 and the methods described in these papers can be adapted for other thiol-modifying photocross-linking reagents. Other RNA molecules contain no thiol groups, and the only primary amines available are on the bases themselves. These groups may be in- volved in Watson-Crick base pairs as part of RNA secondary structures, and these secondary structures may be important for both genetic regula- tion and RNP complex assembly. Amine modification on RNA will, there- fore, not be discussed.

The second method for placing cross-linking groups on RNA involves the use of photoreactive nucleotide analogs. These can be incorporated into the RNA chain during transcription with purified RNA polymerase or in some crude transcription systems. Several types of nucleotide analogs

33 I. Schwartz and J. Ofengand, Proc. Natl. Acad. Sci. U.S.A. 71, 3951 (1982). 34 I. Schwartz, E. Gordon, and J. Ofengand, Biochemistry 14, 2907 (1984).

390 RNA INTERACTIONS [27]

A ,o, ,o, B=A,G, Apu -P-O-CH 2

HO OH

B 0 o II II

N3-K C) k-O-P-O-P-O-CH~ o

HO OH

B = A, ApU

C

o o o Jl II II B = A,G

N3~ C ) )--NH-P-O-P-O-P-O-CH 2 - X ~ / I I ~ Ox,~O,,~ , eo eo eo

'y_._/ HO OH

FIG. I. Initiating photoreactive nucleotide analogs. Three photocross-linking nucleotide analogs that can be placed at the 5' end of a RNA chain are shown. These analogs can be used to initiate transcription with Escherichia coil RNA polymerase. These analogs can be used to probe interactions between proteins and the 5' end of a RNA chain. (A) ct-[(4- Azidophenacyl)thio]nucleotide, (B) fl-(4-azidopbenyl)nucleotide, and (C) y-(4-azidoanili- lyl)nucleotide.

have been characterized, both purines and pyrimidines, with different cross-linking groups, excitation maxima, distances between the nucleo- tide and the cross-linker, and cross-linker position on the nucleotide (base, sugar, or phosphate). The structures of a few of these are shown in Figs. 1-3. Although every photoreactive nucleotide is not shown, repre- sentatives of analogs that can be positioned at the 5' or 3' ends of a RNA chain, or at internal positions within a RNA chain, have been included.

Analogs cannot easily be placed at just one position within a RNA chain. There are exceptions to this, however. Interactions with the 5' end of a RNA molecule can be probed by using an analog that contains the photoreactive cross-linking group on the 5'-phosphate of a mono- or di- nucleotide (Fig. 1). These analogs can be used to initiate transcription, and the cross-linking group will be found only at the 5' end of the RNA chain. Alternatively, one can place a cross-linking group at the 3' end of a

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 391

A 0

0 0 0 HN~ P"~'~N3 / I I J ' l

~O_P_O_P_O_P_O_CH 2~ ,~, ,

HO OH

B S

,

HO OH

C 0 HN..'I~--.,.f 8 r

o o o "~" II _ H il II 0 ' ~ , N 7 ~O-P-O-P-O-P-O-CHo ',~

eo e 0 e 0

HO OH

D 0 I L

0 0 0 HN" ~J "SH II II II 0 ~ . . N /

~O-P-O-P-O-P-O-CH~ ,

HO OH

E o o~-~ c -< ©

n i l II ~O-P-O-P-O-P-O-CH 2

I I I \ - ~ i eO eo e 0

HO OH FIG. 2. Internal photoreactive nucleotide analogs. Five photocross-linking analogs that

can be incorporated at internal positions within RNA chains are shown. All can be used with Escherichia coil RNA polymerase. Some can be used with other polymerases. These ana- logs can be used to probe interactions between proteins and internal sequences of a RNA chain. (A) 5-Azido UTP, (B) 4-thio-UTP, (C) 5-bromo-UTP, (D) 5-mercapto-UTP, and (E) 5'-[(4-azidophenacyl)thio]-UTP.

392 RNA INTERACTIONS [27]

A NHz

0 0 0 N e I ii

O - P - O - P - O - P - O-CHz I I I I \ " J3_ l 0

e 0 eo eo ",~ ~ .~

HO OH

B 0

H N ' ~ , N,~

o o o H N N" 'N e ii ii ii l

O - P - O - P - O - p - O-CH 2 I I I \ - n I

eO eO eo

HO OH

C

0 0 o II II II B = A,G

e O - P - O - P - O - P - O - C H 2 ~,

OH

FIG. 3. Terminating photoreactive nucleotide analogs. Three photocross-linldng analogs that can be incorporated at the Y-terminal position of a RNA chain are shown. All act as RNA chain terminators with Escherichia coil RNA polymerase. These analogs can be used to probe interactions between proteins and the 3' end of a RNA chain. (A) 8-Azido-ATP, (B) 8-azido-GTP, and (C) Y-azido-NTP.

RNA chain by using an analog that functions to terminate transcription (Fig. 3). Cross-linkers can easily be placed specifically at internal posi- tions during transcription only for nucleotides that occur very close to the 5' end of the RNA molecule. While methods for specific placement of nucleotide analogs at internal positions of short RNA molecules have been described, 35,36 these are very complex and not easily applicable to larger RNA chains.

3s M. Krug, P. L. de Haseth, and O. C. Uhlenbeck, Biochemistry 21, 4713 (1982). W. L. Wittenberg and O. C. Uhlenbeck, Biochemistry 24, 2705 (1985).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 393

Even with these limitations, the introduction of photoreactive nucleo- tides into a RNA chain can be used for (1) identification of protein(s) (or other nucleic acids) that interact with or bind to a specific RNA, (2) identification of protein domain(s) or amino acid(s) involved in a specific RNA interaction, and (3) identification of RNA regions or nucleotides involved in a specific protein interaction.

Select ion of Light Source

We have tested several light sources for efficiency of excitation of different photoreactive groups and for effects on protein and nucleic acid structure. These light sources have excitation maxima at 254, 302, or 337 nm. The three light sources used in our studies are (1) 254 nm: short- wavelength mercury vapor lamp (Gates, Long Island, NY), 63/~W/cm 2 at 1 m (from 225 to 313 nm); (2) 302 nm: medium-wavelength mercury vapor lamp (Spectroline, Westbury, NY, model XX-15B), 1800/~W/cm 2 at 15 cm (302 nm maximum); and (3) 337 nm: monochromatic nitrogen laser [Model LN1000, (Photochemical Research Company, Oakridge, TN)], 1300/zJ/pulse, 600-psec pulse, average power at 20 Hz-25 mW.

Light sources were tested for each analog at distances of 5 cm (254 nm), 1.5 cm (302 nm), or 1.0 cm (337 nm). We have found that the 254-nm light source rapidly degrades some proteins in less than a minute, unless samples are shielded. 37 We also see degradation of both DNA and RNA, but degradation occurs much less rapidly. RNA appears to be most stable to irradiation with this light source. Samples have been irradiated with and without cooling, and the degradation is not due to heating. Degrada- tion can be eliminated by irradiating samples in borosilicate tubes, which absorb most wavelengths shorter than 290 nm. Even analogs that have absorption maxima below 290 nm can be activated while shielded, be- cause of the high extinction coefficients of the cross-linking groups; how- ever, longer irradiation times are required when samples are shielded.

The 302- and 337-nm light sources do not degrade protein, DNA, or RNA, even at irradiation times of 30 min. The 302-nm light source does, however, rapidly cleave some disulfide bonds) 7 This cleavage can act to degrade cross-linking reagents, which contain such bonds before covalent attachment of RNA to protein has occurred, or can cause photolytic separation of RNA and protein, which have been joined through the pho- tocross-linking group of disulfide-containing cross-linkers. This disulfide bond breakage is not eliminated by shielding with borosilicate tubes. We

3~ M. M. Hanna, J. Ahdoot, H. Ngyuen, and A. Rafatjoo, unpublished (1987).

394 RNA INTERACTIONS [27]

have found, therefore, that, whenever possible, the 254-nm mercury lamp (with shielding) or the 337-nm laser should be used with reagents having disulfide bonds. If one wishes to sequence proteins and nucleic acids involved in RNP complexes after cross-linking, it is important that a light source that does not degrade these macromolecules be chosen.

S e l e c t i o n a n d S y n t h e s i s o f N u c l e o t i d e A n a l o g

Important considerations for the use of the photoreactive nucleotide analogs shown in Figs. 1-3 will be discussed. Detailed synthetic proce- dures will be given only for those which have not been previously pub- lished, References to the syntheses of the remaining analogs will be pro- vided. Cleavage methods will be described for those analogs that contain cleavable bonds. Figure 1 shows analogs that can be incorporated into the 5' end of a RNA chain, Fig. 2 shows analogs that can be incorporated at internal positions within a RNA chain, and Fig. 3 shows analogs that can be incorporated into the 3' end of a RNA chain.

Initiating Nucleotide Analogs

All of the photoreactive nucleotide analogs shown in Fig. 1 have been tested with Escherichia coli RNA polymerase. None has been tested with SP6, T7, or eukaryotic RNA polymerases, and none is commercially available. These analogs have proved very useful in defining the path of a nascent RNA chain through the E. coli RNA polymerase transcription complex. 38-43 The analogs should also be useful in examining the contacts made with the ribosomal components as the RNA leaves the surface of RNA polymerase.

a-[(4-Azidophenacyl)thio]nucleotides. These nucleotide analogs {5'- [(4-azidophenacyl)thio]phosphorylnucleotides} contain an aryl azide group at approximately the same position as the y-phosphate on a normal nucleoside 5'-trisphosphate. The analogs do not serve as elongation sub- strates for RNA polymerase. Initiation with E. coli RNA polymerase is effective at or above 100/zM, if the normal initiating substrate is kept low (5/zM). If higher concentrations of the normal initiator are needed, the

M. M. Hanna and C. F. Meares, Proc. Natl. Acad. Sci. U.S.A. 80, 4238 (1983). 39 M. M. Hanna and C. F. Meares, Biochemistry 22, 3546 (1983). 4o L. H. DeRiemer and C. F. Meares, Biochemistry 20, 1606 (1981). 4, M. A. Grachev and E. F. Zaychikov, FEBS Lett. 130, 23 (1981). 42 G. A. Nevinski, O. I. Lavrik, O. O. Favorova, and L. L. Kisselev, Bioorg. Khim. 5, 352

(1980). 43 M. A. Grachev, D. G. Knorre, and O. I. Lavrik, Soy. Sci. Rev., Sect. D 2N, 107 (1981).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 395

nucleotide analog concentration should be increased as well. The sulfur- phosphorus bond between the azide and the sugar is stable to acid, base, and thiol, but can be cleaved selectively with organomercurials. This type of cleavable bond is extremely useful, as the bond remains stable through- out transcription, electrophoresis, gel staining and destaining, and most steps involved in the isolation of a cross-linked RNP complex. 38 The azide absorption is at 300 nm (E300,m = 2 × 104 M -l cm-~), but the 337-nm laser is quite effective in its photoexcitation. The 302-nm source can be used, as there are no photolabile disulfide bonds. Synthesis will be described for the mononucleotides. Detailed synthetic procedures for the dinucleotide are given in Ref. 39.

Synthesis of 5'-[(4-Azidophenacyl)thio]phosphoryladenosine or 5'-[(4-Azidophenacyl)thio]phosphorylguanosine

Synthesis should be carried out in dim light. Dissolve adenosine 5'-O- (thiomonophosphate) or guanosine 5'-O-(thiomonophosphate) (Boeh- finger Mannheim, Indianapolis, IN) in water to give a 13 mM solution. Combine 100/zl of this with 10/zl of 0.2 M sodium bicarbonate and 30/~l of methanol. Add to this 30/zl of 90 m M azidophenacyl bromide (Sigma, St. Louis, MO) in methanol and allow the reaction to proceed for 45 min at room temperature. Extract the reaction mixture three times each with isobutyl alcohol and then with ethyl ether and remove the ether under reduced pressure at room temperature. Product can be isolated by anion- exchange chromatography or high-performance liquid chromatography (HPLC), 39 if available. A volatile solvent should be chosen, such as triethylammonium bicarbonate, if using anion-exchange chromatography, so the solvent can be removed by lyophilization. The product should have absorption bands at both 260 nm (from the nucleotide) and 300 nm (from the azide). Product structure can be verified as described in Ref. 39 for the dinucleotide.

Once a RNA chain containing this analog has been cross-linked to a protein (or another nucleic acid) and the RNP complex has been isolated, the two covalently attached molecules can be separated by cleavage of the phosphorus-sulfur bond with phenylmercuric acetate. A saturated solution of phenylmercuric acetate in either water or 0. I% (w/v) sodium dodecyl sulfate (SDS) should be prepared immediately before use. Cleav- age is complete after 24 hr at room temperature, though less time may be required.

fl-(4-Azidophenyl)nucleotides. Analogs of this type have been pre- pared for both adenosine and the dinucleotide adenylyl(3'-5')uridine 5'- phosphate (pApU), and detailed procedures for the synthesis and charac-

396 RNA INTERACTIONS [27]

terization of these are given in Ref. 40. Synthesis occurs by way of a reactive p-azidophenyl phosphorimidazolidate, prepared by the coupling of p-nitrophenyl phosphate with N,N'-carbonyldiimidazole. Since the phosphorimidazolidate reacts with the terminal phosphates of nucleo- sides, synthesis of the guanosine, cytosine, and uridine adducts should also be possible. The absorption maximum for these compounds is at about 260 nm, with an extinction coefficient of E260nm = 2.56 x 104 M -~ cm -1, resulting from the sum of the extinction coefficients for the nucleo- tide and the azide. This type of analog does not contain a chemically cleavable bond.

y-(4-Azidoanililyl)nucleotides. These analogs (nucleoside 5'-triphos- phate y-azidoanilidates) serve both as initiation and elongation substrates for E. coli RNA polymerase41; however, the azide group is cleaved from the analog during incorporation into an internal position in a RNA chain. The azide group, therefore, will remain attached only to the initiating nucleotide. Synthesis of the adenosine, guanosine, and cytosine analogs have been described. 42,43 The absorption maximum for these compounds is at 260 nm. The nitrogen-phosphorus bond linking the azide and the sugar is acid-labile. This allows covalently attached RNA and protein molecules to be separated by treatment with 10% (v/v) acetic acid (2 hr at 37°). The major disadvantage of this type of analog is the instability of the RNA-protein cross-link to standard protein staining and destaining pro- cedures, as acetic acid is frequently used to fix proteins and RNA in gels.

Internal Nucleotide Analogs

The photoreactive nucleotide analogs shown in Fig. 2 have all been incorporated into internal positions in RNA chains with E. coli RNA polymerase. Some have also been used with SP6, T7, or eukaryotic RNA polymerases. Only 5-bromo-UTP is commercially available (Sigma).

4-Thiouridine 5'-Triphosphate. The thioketo groups of thiopyrimidine derivatives can be modified with photoreactive cross-linking groups or can be directly activated with long-wavelength UV light to induce cross- linking. 4-Thiouridine and its corresponding nucleotides undergo phot 0- oxidation to electrophilic intermediates that react with nucleophiles under mild conditions. 44,45 As the cross-linking group is part of the pyrimidine, 4-thiouridine can be used to examine contacts with RNA within a few angstroms of uridine residues. The excitation maximum for this analog is between 320 and 340 nm. The nitrogen laser is an excellent photoexcita- tion source37; however, the 302-rim mercury lamp can be used as well because of the absence of disulfide bonds. 4-Thioufidine has been shown

44 M. Pleiss, H. Ochiai, and P. A. Cerutti, Biochem. Biophys. Res. Commun. 34, 70 (1969). 45 M. G. Pleiss and P. A. Cerutti, Biochemistry 10, 3093 (1971).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 397

to be taken up and converted to a substrate for both prokaryotic 46 and eukaryotic RNA polymerases 47 in vivo. 4-Thio-UTP has been shown to be a substrate for HeLa RNA polymerase 11, 48 T7, 49 E. co l i : ° and SP651 RNA polymerases in vitro.

While the original synthesis of 4-thio-UTP was chemical, 52 the avail- ability of 4-thio-UDP (Sigma) allows for very simple and rapid enzymatic synthesis. A method involving transfer of a y-phosphate from ATP using nucleoside diphosphate kinase has been published. 48 This method, how- ever, results in the copurification of some ATP with the 4-thio-UTP. This can be a problem for some strategies used to place cross-linking groups at just one position in a RNA chain. An alternate synthesis that allows for the purification of 4-thio-UTP, which is free from all other nucleotides, involves the addition of a y-phosphate using glycolytic enzymes. 49

Synthesis of 4-Thiouridine 5'-Triphosphate

Synthesis is described for the preparation of 0.5 tzmol of 4-thio-UTP; however, this synthesis has been scaled up 10-fold with similar yields. All steps should be carried out on ice or at 4 ° , unless otherwise specified. Sodium pyruvate and cysteine solutions must be prepared immediately before use. Aqueous solutions of L-a-glycerol phosphate and/3-NAD can be stored at -20 ° for 6 months.

Prepare an ammonium sulfate suspension of enzymes (see Enzyme Stock) containing the following: 0.1 mg of glycerol-3-phosphate dehydro- genase, 0.001 mg of triose-phosphate isomerase, 0.1 mg of glyceralde- hyde-3-phosphate dehydrogenase, 0.01 mg of 3-phosphogiycerate kinase, and 0.05 mg of lactate dehydrogenase. Enzymes can be purchased from Boehringer Mannheim as ammonium sulfate suspensions and should be mixed gently before use. Spin the enzyme solution for 5 rain in a micro- centrifuge and remove the supernatant solution by aspiration. The en- zyme pellet should be stored on ice until just before use. Prepare a 60 mM cysteine stock solution, pH 8-9, by dissolving l0 mg of cysteine hydro- chloride and 12.5 mg of Tris base in 1.0 ml of water. Prepare an enzyme- cysteine mixture (solution A) by resuspending the enzyme pellet in 0.333

46 E. Hajnsdorf, A. Favre, and A. Expert-Bezanqon, Nucleic Acid Res. 14, 4009 (1986). 47 W. T. Melvin, H. B. Milne, A. A. Slater, H. J. Allen, and H. M. Keir, Eur. J. Biochem.

92, 373 (1978). '~ B. Bartholomew, M. E. Dahmus, and C. F. Meares, J. Biol. Chem. 261, 14226 (1986). ~9 N. K. Tanner, M. M. Hanna, and J. Abelson, Biochemistry 27, 8852 (1988). 5o F. Cramer, E. M. Gottschalk, H. Matzura, K.-H. Scheit, and H. Sternbach, Eur. J.

Biochem. 19, 379 (1971). 51 M. M. Hanna, unpublished (1987). 52 K. H. Scheit, Chem. Ber. 101, 1147 (1968).

398 RNA INTERACTIONS [27]

ml of 0.15 M Tris-HCl, pH 9.0, and then by adding 0.667 ml of 60 m M cysteine stock. This solution should be stored on ice until needed.

~prepare a 40 m M sodium pyruvate solution by placing approximately 5 m g o f sodium pyruvate in a dry test tube and by dissolving in water to give a 4 ~ m g / m l solution. All remaining steps should be carded out in dim light. Prepare 3.5 ml of solution B, as shown below. Add 0.875 ml of solution A and'-4.4 ml of 0.6 m M phosphoric acid (HPLC grade) to solu- tion B. Incubate ~q.e mixture for l hr at 25 ° and then extract twice with phenol to remove enzymes before purification.

Dilute the reaction mixture to 100 ml with water and load onto a 1.75 x 25-cm Whatman DE-52 column (at 4°), equilibrated in 0.1 M triethylam- monium bicarbonate (TEAB), pH 8.0. Wash the column with 10 column volumes of 0.1 M TEAB and elute the product with a linear gradient from 0.5 to 1.0 M TEAB, pH 8.0 (500 ml of each). 4-Thio-UTP elutes at approx- imately 0.7 M. Locate the product fractions by determining absorbance at 330 nm, pool, and lyophilize to remove the solvent. The product should be dissolved in water and again ]yophilized two or three times or until all visible salt is removed. Dissolve the product in water and determine the concentration by dilution of an aliquot into 0.01 M phosphate buffer, pH 7, using an extinction coefficient of E330nm = 21.2 x 103. 53 If the product has been dried for a long time and resists dissolution, 2-mercaptoethanol can be added on a molar basis to the 4-thio-UTP.

Enzyme Stock

Enzyme Stock Volume

Glycerol-3-phosphate dehydrogenase Triose-phosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase 3-Phosphoglycerate kinase Lactate dehydrogenase

I0.0 mg/ml 10.0/~1 2.0 mg/rnl 0.5/.d

10.0 mg/ml 10.0 pl 10.0 mg/ml 1.0 pl 5.0 mg/ml 10.0 pl

Solution B

Component Final concentration Stock concentration Volume/3.5 ml

Tris-C1, pH 9 0.15M 0.5 M 1.05 ml D'l~ 0.015 M 1.0 M 0.052 ml Magnesium chloride 0.035 M 1.0 M 0.122 ml c~-Glycerophosphate 0.34 mM 0.02 M 0.057 ml 4-Thio-UDP 0.14 mM 0.1 M 0.005 ml fl-NAD 1.43 mM 0.035 M 0.143 ml Sodium pyruvate 2.8 mM 0.04 M 0.245 ml Water 1.82 ml

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 399

5-Mercaptouridine 5'-Triphosphate. While 5-mercapto-UTP (Fig. 2) does not contain a highly photoreactive group, 5-mercapto-UTP does contain a thiol group, which can be modified with thiol-specific bifunc- tional photocross-linking reagents (Table I). 5-Mercapto-UTP is an excel- lent substrate for E. coli RNA polymerase) ~ Since the 5 position of the uracil is not involved in normal Watson-Crick base pairing, RNA con- taining this analog should form normal secondary and tertiary structures, even at high levels of substitution. RNA, containing complete substitution of 5-mercapto-UTP for UTP, forms normal p-independent transcription termination signals, as recognized by E. coli RNA polymerase. 5~ Analog- containing RNA can be posttranscriptionally modified to place photo- cross-linking groups on accessible thiol groups. Residues that are accessi- ble to these reagents are most likely to be accessible to regulatory or structural proteins as well. Even without posttranscriptional modifica- tion, there is some photocross-linking of 5-mercapto-UTP-containing RNA (not seen with UTP-containing RNA), when irradiated with long- wavelength UV light (wavelengths longer than 300 nm).

Synthesis of 5-Mercaptouridine 5'-Triphosphate

5-Mercapto-UTP is synthesized by a modification of the synthetic procedure for 5-mercaptouraciP 4 and 5-mercapto-UMP) 5 Fresh reagents must be used. Dissolve 2.0 grams (4 mmol) of UTP in 125 ml of dimethyl- acetamide (4 °) in a 500-ml-stoppered Erlenmeyer flask. Stir at 4 ° for at least 4 hr. A suspension of UTP will result. Prepare a fresh solution of methyl hypobromite 56 by placing 75 ml of anhydrous methanol (HPLC grade, -20 °) into a 250-ml Erlenmeyer flask, containing a magnetic stir bar and capped with a stopper. Insert a drying tube and a 5-ml syringe (16- to 18-gauge needle) through holes in the stopper. Place the flask in a dry ice-ethanol bath to maintain the temperature at or below - 15 ° and place the apparatus in dim light. Add 28.0 g (100 mmol) of silver carbonate and stir vigorously for 5 min. The solution should turn an olive-green color. While maintaining the temperature below -15 ° and stirring vigorously, add 5.5 ml (100 mmol) of bromine dropwise via the syringe at a constant rate of 1 ml/min. Stir this solution for 4 hr in the dark, maintaining the temperature below -15 °. The color will change to a yellowish green.

53 M. Pleiss, H. Ochiai, and P. A. Cerutti, Biochem. Biophys. Res. Commun. 34, 70 (1969). 54 T. J. Bardos and T. I. Kalman, J. Pharm. Sci. 55, 606 (1966). ~5 y . K. Ho, L. Novak, and T. J. Bardos, in "Nucleic Acid Chemistry" (L. B. Townsend

and R. S. Tipson, eds.), Vol. 2, p. 813. Wiley, New York, 1976. 56 R. Duschinsky, T. Gabriel, W. Tautz, A. Nussbaum, M. Hoffer, E. Grundberg, J. H.

Burchenal, and J. J. Fox, J. Med. Chem. 10, 47 (1967).

400 RNA INTERACTIONS [27]

While the above solutions are stirring, prepare a fresh solution of sodium disulfide by dissolving 23 g (96 retool) of sodium sulfide nonahy- drate in 150 ml of boiling 100% (v/v) ethanol in a 250-ml beaker. Add 3.1 g (96 mmol) of sulfur and continue stirring until the volume is less than about 25 ml. This requires about 1 hr. Do not heat longer than needed. Cool the solution in an ice bath and a yellow solid will form. This solid must be broken up into small pieces as the solid forms to prevent forma- tion of a large, insoluble solid. Store under vacuum and on dry ice until needed.

After the methyl hypobromite solution has stirred for 4 hr, filter the solution through Celite or through a medium glass-fritted funnel into a cold and dry 125-ml filtration flask, maintaining the temperature below -15 ° with dry ice. Rinse the flask, which contained the methyl hypobro- mite, four times each with 2 ml of dimethylacetamide and wash the filter with these rinses. Immediately add this solution to the suspension of UTP dimethylacetamide. A yellow slurry will form. Cover the reaction mixture with foil and continue stirring vigorously at 4 ° for 5-6 hr. The reaction mixture should change to a clear orange and should be used immediately. Add 23 g (80 mmol) of the freshly prepared sodium disulfide to the clear orange solution. Stir for at least 12 hr at 4 °, in the dark, or until the solution turns and remains dark brown (no longer than 48 hr). The inter- mediate product bis(uridine 5'-triphosphate-5-yl) disulfide (bis-UTP) can be stored at 4 ° in the dark at this stage, if necessary, before removal of other reactants and side products.

Purify bis-UTP by adding 1 liter of cold water to the reaction mixture and by extracting three times each with 250 ml of water-saturated diethyl ether. A small amount of precipitate will form. Filter the aqueous phase first through Whatman 1 filter paper and then through a 0.2-/zM filter. Wash the precipitate with water, combine the filtrate and water, and wash and dilute with water until the conductivity is equal to or below that of 0.02 M TEAB, pH 8.3, at room temperature (several liters). Load this onto a 1200-ml DE-52 column (at room temperature), equilibrated in 0.02 M TEAB, pH 8.3. Wash the column with 5 column volumes of 0.02 M TEAB and then elute the product with a 4-liter linear gradient from 0.02 to 1.5 M TEAB, pH 8.3. Locate bis-UTP by monitoring absorbance at 273 nm. The disulfide, containing bis-UTP, absorbs at 273 nm. When the disulfide bond is reduced, the 5-mercapto-UTP formed will also adsorb at 330 nm (at pH 8).

Pool the product fractions and remove the solvent under vacuum. Lyophilization for 3-5 days, with several reconstitutions with DEPC- treated water, is sufficient to remove salt. 5-Mercapto-UTP can be pre- pared for use in transcription by reduction of bis-UTP with DTT. Adjust

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-VROTEIN 401

the solution to pH 8 and determine the yield using E330n m = 8 )< 10 3 M -1 cm -~ for 5-mercapto-UTP. 54,55

5-Azidouridine 5'-Triphosphate. This UTP analog contains an azide group on the 5 position of the uridine ring (Fig. 2). Modification at this position should not interfere with formation of normal base pairs with other nucleotides. 5-Azido-UTP synthesis is described in Ref. 57. This analog is a substrate for E. coli RNA polymerase and has been used to photoaffinity label the active site of this enzyme in a ternary transcription complex) s The excitation maximum for this analog is 288 nm.

5-[(4-Azidophenacyl)thio]uridine 5'-Triphosphate. This UTP analog contains an aryl azide group about 10 A from the 5 position of the uridine residue (Fig. 2), which should not interfere with normal Watson-Crick base pairing. The azide absorption is at 300 n m (E300nm -= 2 × 104 M -1 cm-l), and the 302-nm light may be used, because there are no disulfide bonds present. This analog is synthesized by the alkylation of 5-mercapto- UTP (5-SH-UTP) with azidophenacyl bromide) 9 The sulfur atom be- tween the nucleotide and the azide can be removed by desulfurization with Raney nickel with accompanying bond scission, making this a re- versible cross-linker. The sulfur-carbon bonds between the azide and the base are stable to acid, base, and thiol, however. This type of cleavable bond is extremely useful, as the bond remains stable throughout transcrip- tion, electrophoresis, gel staining and destaining, and most steps involved in the isolation of a cross-linked RNP complex. The analog can be incor- porated into internal positions in RNA molecules by E. coli RNA poly- merase and has been used to identify the contacts between this enzyme and the nascent RNA during transcription in vitro. 6°

Terminating Nucleotide Analogs

The photoreactive nucleotide analogs shown in Fig. 3 cannot be incor- porated into internal positions in a RNA chain by E. coli RNA poly- merase. Their use with other RNA polymerases has not been examined. These terminating analogs can be used to probe the active site of a RNA polymerase, as phosphodiester bond formation occurs at the 3' end of the RNA.

8-Azidonucleoside 5'-Triphosphates. The 8-azidopurines act as RNA

57 R. K. Evans and B. E. Haley, Biochemistry 26, 269 (1987). 5a A-Y. M. Woody, R. K. Evans, and R. W. Woody, Biochem. Biophys. Res. Commun. 150,

917 (1988). 59 M. M. Hanna, S. Dissinger, B. D. Williams, and J. E. Colston, Biochemistry, in press

(1989). 6o S. Dissinger and M. M. Hanna, in preparation.

402 RNA INTERACTIONS [27]

chain terminators, because the azide group on position 8 forces the nucle- otide into a conformation that does not allow further chain elongation. Whether these analogs would act as terminators with other RNA poly- merases is not known. The synthesis of 8-azido-ATP (Fig. 3) from 8- bromo-ATP is described in Ref. 61. This analog is also commercially available from Sigma. This analog has an absorption maximum at 281 nm at pH 7.4 with E 2 s Inm = 1.33 x 104 M -1 cm-l. 61 The GTP analog can be synthesized in a similar manner. These compounds are stable, when stored in methanol at - 2 0 ° (or lower), showing 5-10% decomposition in 5-6 months. Samples can be photolyzed at 254 nm for times ranging from 15 sec to 5 min, depending on the intensity of the light source and the distance of the sample from the light source. 62 Irradiation should be done with shielding if proteins are to be analyzed. We have found, however, that both the 302- and 337-nm light sources are more effective for activa- tion of 8-azido-ATP than the 254-nm light source, without the need for shielding to protect proteins and nucleic acids) 7 Detailed procedures for the use of 8-azidopurine analogs has been discussed in detail in an earlier volume of this series. 62

Y-Azidonucleoside Triphosphates. The 3'-azidonucleotides (Fig. 3) act to inhibit transcription, because there is no hydroxyl group on the 3' position of the ribose to allow phosphodiester bond formation with the next nucleotide in the RNA chain. The syntheses of both the purine analogs are described in Ref. 63. These analogs should cause termination of transcription with all RNA polymerases for which these analogs may be substrates, similarly to other 3'-substituted ribonucleotides. 64 This type of analog allows one to examine the contacts made with the ribose in the active site of RNA polymerases. 65

RNA-Protein Cross-Linking with Photoreactive Nueleotide Analogs

Synthesis of Analog-Containing RNA

All of the nucleotide analogs shown in Figs. 1-3 contain 5'-triphos- phates and are best incorporated into RNA chains via transcription in the absence of light. Radioactivity can be incorporated into the RNA at inter- nal positions with c~-32p-labeled nucleotides. If the analog-containing RNA will be sequenced after cross-linking to protein, the 5' end of the

61 B. E. Haley and J. F. Hoffman, Proc. Natl. Acad. Sci. U.S.A. 71, 3367 (1974). 62 R. L. Potter and B. E. Haley, this series, Vol. 91, p. 613. 63 D. Panka and D. Dennis, J. Biol. Chem. 259, 8384 (1984). 64 H. T. Shigeura and G. E. Boxer, Biochem. Biophys. Res. Commun. 17, 758 (1964).

V. W. Armstrong and F. Eckstein, Biochemistry 18, 5117 (1979).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 403

RNA can be labeled by incorporation of y-32p-labeled nucleotides (if a photoreactive-initiating nucleotide analog is not being used). Alterna- tively, one can initiate transcription with a dinucleotide, which does not contain a 5'-phosphate, and can add radioactivity with T4 polynucleotide kinase after isolation of the RNA. If a 5' analog is used, the RNA can be end labeled at the 3' end with RNA ligase. By making the RNA radioac- tive, one can identify proteins that become covalently attached to the RNA by cross-linking during irradiation, thereby becoming radioactively labeled.

Although all of the analogs shown are substrates for E. coli RNA polymerase, only 4-thio-UTP has been tested with SP6, T7, and eukary- otic RNA polymerases. It is probable that the uridine analogs substituted in the 5 position will be substrates for at least T7 RNA polymerase, as these analogs contain groups that are smaller than those found in biotiny- lated UTP, which does serve as a substrate for this enzyme. 66

With the exception of 4-thio-UTP and 5-bromo-UTP, all of the analogs shown contain azide groups. Because azides are rapidly reduced by com- pounds containing two thiol groups, such a s DTT, 67 one must eliminate such compounds from the transcription buffer and try to keep the pH of the solution below 8.0. Monothiols will also reduce azides, but less rap- idly. It is therefore desirable to replace DTT with a monothiol, such as 2- mercaptoethanol, if thiol is absolutely required in the reaction. We have found, however, that thiol can be eliminated from the transcription buffer for E. coli RNA polymerase with no loss of activity for several hours.68 It is also possible to completely remove thiol from the E. coli RNA poly- merase storage buffer with no loss of transcriptional activity or specificity for up to 2 weeks, when the enzyme is stored at -20 °.

In order to study interactions between RNA and RNA polymerase or transcription factors, it is not necessary to isolate the analog-containing RNA before cross-linking. Transcription complexes can be irradiated dur- ing active transcription, or transcription can be stopped without dissocia- tion of the transcription complex by incorporation of terminating nucleo- tides. 3s To examine interactions between analog-containing RNA and proteins not involved in transcription, it is possible, in some cases, to add the purified protein or protein-containing fraction directly to the transcrip- tion reaction after synthesis of the RNA is complete. One must judge each system individually when deciding on an approach. It is frequently neces-

~6 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981).

6~ j. V. Staros, H. Bayley, D. N. Standring, and J. R. Knowles, Biochem. Biophys. Res. Commun. 80, 568 (1978). M. M. Hanna, C. Bowser, and M. Hodge, unpublished (1987).

404 RNA INTERACTIONS [27]

sary to isolate the RNA and then to add the RNA back to purified proteins or extracts containing these proteins. Most of the methods described below are applicable to all of these approaches.

Isolation of Analog-Containing RNA

All steps must be carried out in dim light. There are several ways in which RNA can be isolated. The method chosen may depend on the importance of the RNA secondary and tertiary structure to the RNA- protein interaction of interest. When working with a large molecule with extended secondary structure, it may be desirable to choose an isolation method that does not fully denature the RNA, as the correct structure may not reform after isolation. We have found the following methods to be most useful.

Precipitation ofRNA. If working with a system that allows the prepa- ration of analog-containing RNA with purified DNA and RNA poly- merase where only one RNA transcript is formed, it is possible to simply remove the polymerase and DNA from such reactions. After transcrip- tion, the DNA template can be degraded by treatment of the reaction mixture with DNase, which is free of RNase (RQI DNase, Promega, Madison, WI). RNA polymerase and the DNase can be removed by ex- traction of the transcription reaction with phenol. The RNA can be pre- cipitated two times with ammonium acetate-ethanol to remove unincor- porated nucleotides 69 and can be resuspended in the appropriate buffer for formation of the RNP complex. (This buffer should not contain dithiols, if an azide cross-linker is being used.)

Isolation by Gel Electrophoresis. RNA can be isolated on nondena- turing or denaturing acrylamide or agarose gels, depending on the size of the RNA. Gels should be prepared and run according to standard proce- dures. 69 After electrophoresis, the radioactive RNA can be located by autoradiography (gels should not be fixed, stained, or dried). It is impor- tant to place radioactive marker dots or light spots all around the gel so that, after development, the film can be aligned accurately above the gel to allow excision of the RNA. If the RNA is very radioactive, one can expose the gel to film at room temperature. However, sometimes gels will need longer exposures, making freezing the gel and use of an intensifying screen desirable. When working with agarose or very low-percentage acrylamide gels (less than 10%), freezing and thawing of the gel can cause the gel to change size and become "mushy." We have had the best results by leaving the gels on one glass plate and by prefreezing the gel on a slab

69 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Man- ual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 405

of dry ice before putting film on the gel. It is best to use two pieces of X-ray film, saving one as a record of the results of the experiment and using the other to directly overlay on the gel. It is easiest to cut directly through this second piece of film and the underlying gel with a razor blade. It is important that the gel remain frozen through all steps, again being placed on dry ice, while the gel piece is being cut from the gel.

The RNA can be recovered from the gel slice by one of several meth- ods. For small RNA molecules, the gel piece can be crushed, and the RNA can be isolated by diffusion. An example for the isolation of analog- containing tRNA is given in Ref. 49. Alternatively, one can place the gel piece in a dialysis bag and isolate the RNA by electrophoresis. This is effective even for large RNA molecules and RNA-protein complexes. An example of this method is given in Ref. 38. Similarly, the RNA can be isolated by electrophoresis into a high-salt solution using an electroeluter apparatus (IBI, New Haven, CN). Last, dissolvable gels, which will be described in a later section, can be used.

Isolation by Affinity Chromatography. RNA can be separated from protein and DNA by passage over small columns containing covalently linked borate g r o u p s . 70-73 These groups have affinity for the cis-diol on the 3'-terminal ribose of RNA. Columns contain acetylated N-[N'-(m-di- hydroxyborylphenyl)succinamyl]aminoethyl cellulose (DBAE-cellulose), available from Collaborative Research (Waltham, MA), or DBASE- polyacrylamide (Affi-Gel 601), available from Bio-Rad (Richmond, CA). RNA can be eluted from these columns with 0.05 M sodium acetate, pH 5.1, containing 0.2 M NaC1 and 1 mM EDTA, and can be directly precipi- tated with ethanol. With proper storage, these columns can be regener- ated and reused up to 60 times. 73

Preparation of RNA-Protein Cross-Linked Complexes

Once a cross-linking reagent and a light source have been chosen, one must determine the optimal time for cross-linking empirically. If the RNA and protein associate to form a stable, long-lived complex, it is possible to irradiate for very long times and get cross-linking yields as high as 90%. If one is examining a transient interaction, however, it may be necessary to irradiate for shorter times, usually giving much lower cross-linking yields. It has recently been shown that salt can greatly affect the strength of protein-nucleic acid complexes. Substitution of glutamate for chloride ions has increased protein-DNA interactions more than 10-fold in some

70 H. L. Weith, J. L. Wiebers, and P. T. Gilham, Biochemistry 9, 4396 (1970). 7s R. K. Goitein and S. M. Parsons, Anal. Biochem. 87, 636 (1978). 72 A. E. Annamalai, P. K. Pal, and R. F. Colman, Anal. Biochem. 99, 85 (1979). 73 S. Ackerman, B. Cool, and J. J. Furth, Anal. Biochem. 100, 174 (1979).

406 RNA INTERACTIONS [27]

cases. TM It may be worthwhile to optimize the salt concentration for each RNP complex before photocross-linking.

There are several controls which must be included to ensure that photocross-linking data are meaningful. These include the following.

1. Always analyze a sample that has been treated identically to the photolyzed sample, except for irradiation. These dark controls are criti- cal, as radioactively labeled nucleotides can become covalently attached to proteins nonenzymatically, even in the absence of light. 75 Although this labeling occurs at a low efficiency, this labeling can be of the same order of magnitude as some photocross-linking.

2. Set up parallel cross-linking reactions with a protein of similar size and charge, as the protein proposed to be involved in the RNP complex. This will help one to distinguish between specific and nonspecific binding to RNA.

3. Set up parallel cross-linking reactions with a RNA of similar size and secondary structure, if possible, as the RNA proposed to be involved in the RNP, for the same reason mentioned previously.

Isolation and Analysis of Cross-Linked RNA-Protein Complexes

In the simplest case, one may simply wish to ask whether a certain RNA and protein interact. This is done most easily by analyzing the cross-linking reactions (including controls) by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography. As previously mentioned, if the RNA is radioactive and the protein is not, the formation of a radio- active protein molecule during irradiation of the RNP complex indicates an interaction between the RNA and the protein. Using electrophoresis, cross-linked complexes can be separated from free RNA and protein. A problem arises with large RNA molecules and small proteins, as both will tend to comigrate in these gels. The following method is useful for separa- tion of free RNA from protein and RNA-protein complexes. It involves the simultaneous electrophoretic transfer of these molecules from the gel to overlayed nitrocellulose and nylon membranes, which can then be probed with antibodies or can be subjected to staining and autoradiogra- phy. Proteins and RNA-protein complexes will stick to the nitrocellulose and free RNA will bind to the nylon.

Transfer of Cross-Linked RNA-Protein Complexes to Nitrocellulose and Nylon Membranes

Unless otherwise stated, all procedures should be carried out in the dark with only a red light. All paper and membranes should be wetted in

74 s. Leirmo, C. Harrison, D. S. Cayley, R. R. Burgess, and M T. Record, Jr., Biochemistry 26, 2095 (1987).

75 M. C. Schmidt and M. M. Hanna, FEBS Lett. 194, 305 (1986).

[ 2 7 1 PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 407

transfer buffer (25 mM Tris, 192 mM glycine, and 20% (v/v) methanol). Tris-glycine solution should be prepared in DEPC-treated water and should be autoclaved before HPLC-grade methanol is added. Transfer buffers can be varied to favor transfer of a given RNA-protein complex. After separating components of the cross-linking reaction(s) by gel elec- trophoresis, transfer the gel from the glass plate to Whatman chromatog- raphy paper (Whatman 3MM). Place the gel on an electroblot apparatus (as used for western transfers). Layer onto the gel in the following order: a piece of Biotrace NT nitrocellulose [#66486S (Gelman Sciences, Ann Arbor, MI)], a piece of chromatography paper, a piece of Nytran nylon membrane [0.2-/zm pore size (Schleicher and Schueller, Keen, NH)], and another piece of chromatography paper. Make sure there are no air bub- bles between the layers. Enclose the layered sandwich of paper-gel- nitrocellulose-paper-nylon-paper in two larger, folded pieces of chroma- tography paper. Close the cassette assembly and place the cassette vertically in the blotting unit in the following orientation:negative elec- trode (black), black Scotch-Brite pad, paper, gel, nitrocellulose, paper, nylon, paper, white open-cell sponge pad, and positive electrode (red). Transfer should be carried out in the dark at 4 ° for 12-48 hr at 150 mA. Transfer is carded out in the dark so that photoreactive molecules, which may not have been activated during photolysis or are in the control reac- tions, will not become covalently linked to the gel or the membranes by room light during transfer.

After transfer, procedures can be carded out with the lights on. Re- move the cassette assembly from the blotting unit and air dry the nylon membrane between Kimwipes and paper towels. Nitrocellulose can be probed with antibody against protein according to standard procedures. Both the nitrocellulose and the nylon membranes can then be subjected to autoradiography to locate RNA-containing bands. RNA-protein com- plexes should be bound to the nitrocellulose. Free RNA should be bound to the nylon. The gel should also be analyzed by staining and autoradiog- raphy to ensure that complete transfer has occurred. If a radioactive band is present on the nitrocellulose in the same place as a band visualized with antibody, then the band is inferred to correspond to a protein-RNA cross-linked complex.

Isolation of Cross-Linked RNA-Protein Complexes

To determine which domains or regions of a protein or RNA molecule, or more specifically, which amino acids or nucleotides, are involved in a RNA-protein complex, the covalently linked RNA-protein complex must be separated from free RNA and protein before beginning character- ization of the cross-linked molecules. If antibody to the protein involved

408 RNA INTERACTIONS [27]

in the complex is available, the RNA-protein complex, along with free protein, can be separated from free RNA and other proteins in the reac- tion by immunoprecipitation. The free protein in the immunoprecipitated fraction can then be separated from the RNA-protein complex using a borate column, as described previously. 7°-73

Alternately, cross-linked RNA-protein complexes can be isolated by gel electrophoresis. When cross-linkers have been used which do not contain disulfide bonds, standard SDS-polyacrylamide gels containing methylenebisacrylamide can be used , 76 including thiol in the sample load- ing buffer. For very large RNA molecules cross-linked to small proteins, there is sometimes a problem in separating free RNA from RNA-protein complexes on such gels. Better resolution can sometimes be obtained by using polyacrylamide-urea gels. 77 If the two still cannot be separated, it may be necessary to partially digest the R N A before attempting separa- tion. This will result in a small piece of RNA cross-linked to the protein, and the protein size will dictate mobility in these gels. When cross-linkers have been used which contain disulfide bonds, cross-linked proteins can be isolated on thiol-dissolvable acrylamide gels containing bisacrylcyst- amine.TS-S0

When reaction components have been separated by gel electrophore- sis, it is necessary to recover the RNA-protein complex from the gel for further analysis. Gels should not be stained, destained, or dried after electrophoresis. It is advisable to run marker proteins in one lane of the gel, which can be sliced offfor staining and destaining, to approximate the position of the proteins of interest in the unstained portion of the gel. RNA-protein complexes are located by autoradiography of the gel, as described in Isolation of Analog-Containing RNA. One must identify the band which is believed to correspond to the cross-linked complex by comparing the photolyzed test reaction to all of the control reactions. The RNA-protein complex can be recovered from the gel piece by any of the methods described in Isolation of Analog-Containing RNA.

Analysis o f Cross-Linked R N A - P r o t e i n Complexes

Several questions can be answered once the RNA-protein complex has been isolated. If antibody to the protein is available, the identity of the protein(s) in such a complex can be determined or verified by the methods

76 U. Laemmli, Nature (London) 277, 680 (1970). 77 C. W. Wu, F. V. H. Wu, and D. C. Speckhard, Biochemistry 20, 5449 (1977). 78 j, N. Hansen, Anal. Biochem. 76, 37 (1976). 79 j. N. Hansen, B. H. Pheiffer, and J. A. Boehnert, Anal. Biochem. 105, 192 (1980). so j. N. Hansen, Anal. Biochem. 116, 146 (1981).

[27] PHOTOAFFINITY CROSS-LINKING OF RNA-PROTEIN 409

described previously. If antibodies are not available, the RNA can be cleaved from the protein, and both can be analyzed by gel electrophoresis or some other physical method for which characteristics of the putative cross-linked RNA or protein are known. If a cross-linker which contains a cleavable bond has been used, the RNA and the protein(s) can be sepa- rated simply by incubation of the complex with the appropriate cleavage reagent. If there is no cleavage procedure to separate the RNA and the protein, the RNA can be digested with ribonucleases or by treatment with alkali to leave a tag on the protein. Similarly, the protein can be digested with proteases, leaving a tag on the RNA.

To identify domains of the RNA and/or protein that are involved in the cross-link, RNA and/or protein sequencing must be used. If the RNA has been radioactively end labeled, the RNA cross-linked to protein can be sequenced by chemical sLs2 or enzymatic s~,84 means and can be compared to the sequencing pattern for RNA that has not been cross-linked. Se- quencing patterns for both RNAs will be identical for all nucleotides between the radioactive label (whether 5' or 3') and the nucleotide that is cross-linked to protein. The nucleotide that is cross-linked to protein and all nucleotides beyond the bound protein and the labeled end will have an aberrant mobility on sequencing gels and will be missing from the se- quencing ladders. If the RNA on the RNA-protein complex was partially digested to facilitate isolation of the cross-linked complex, the RNA can be relabeled by treatment with phosphatase to remove the 5'-phosphate group and by addition of a [32p]phosphate group with polynucleotide kinase. 69

To identify the domains of a protein that are involved in a cross-link, it is best to use uniformly labeled, whole-body [t~-32p]RNA for cross-link- ing. After isolation of the complex, the RNA can be digested, preferably chemically, so that only small pieces of radioactive RNA remain attached to the protein. This complex can then be treated with proteases, and the radioactively labeled fragment can be isolated and identified by standard protein-sequencing techniques.

A c k n o w l e d g m e n t s

Work from this laboratory was supported in part by an American Cancer Society grant (#NP544A) and by University of California Cancer Research Coordinating Committee funds.

at D. A. Peattie and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 77, 4679 (1980). u D. A. Peattie, Proc. Natl. Acad. Sci. U.S.A. 76, 1760 (1979). s3 H. Donis-Keller, A. M. Maxam, and W. Gilbert, Nucleic Acid Res. 4, 2527 (1977). 84 H. Donis-Keller, Nucleic Acid Res. 8, 3188 (1980).