acyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP.
('.-D 1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 9 2073 -2079
Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E.coli and T7 RNA polymerases Michelle M.Hanna, Yuying Zhang, Jack C.Reidling*, Matthew J.Thomas+ and Jerry Jou Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Rm. 135, Norman, OK 73019, USA Received February 22, 1993; Accepted March 29, 1993
ABSTRACT A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E.coll and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APASCMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E.coli RNA polymerase, downstream of the X PR' promoter (+ 16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin.
INTRODUCTION Specific RNA-protein and RNA-nucleic acid interactions play important roles in the formation of stable structural components of cells, such as ribosomes and small nuclear ribonucleoprotein particles. Additionally, these interactions are involved in many stages of gene expression and protein synthesis, playing both regulatory and catalytic roles in transcription, RNA processing, splicing, translation and protein targeting. Identification of the specific molecular interactions involved provides insight into the mechanisms of these processes and contributes to an understanding of the general parameters of molecular recognition involving nucleic acids. Photochemical crosslinking is a powerful *
To whom
tool for the identification of the specific interactions involved in RNA-protein and RNA-nucleic acid complexes. One approach involves incorporation of nucleotide analogs containing photoreactive crosslinking groups into the RNA. RNA-protein or RNA-nucleic acid complexes are then formed, the complex is irradiated with ultraviolet (UV) light, and molecules covalently attached to the RNA by photocrosslinking are identified. The use of such analogs results in much higher RNA crosslinking yields than direct crosslinking by excitation of unmodified nucleic acids. Nucleotide analogs have been described that can be incorporated enzymatically by RNA polymerases specifically into the 5' end, 3' end, or internally in the RNA transcript (for review, see ref. 1). We have also placed crosslinking groups at specific internal UMP residues in RNA utilizing automated chemical synthesis and a protected phosphoramidite precursor (2). Until now, placement of photocrosslinking analogs internally into RNA enzymatically, utilizing in vitro transcription systems, has also been limited to uridine analogs. One of these analogs, 5-((4-azidophenacyl)thio)-UTP (5-APAS-UTP) (3), which contains a photoreactive aryl azide group approximately 10 A from the base, is incorporated at internal positions in RNA by both E. coli and T7 RNA polymerases, without interfering with normal Watson-Crick base pairing (4). Other UTP analogs that are incorporated internally during transcription include 4-thioUTP, 5-bromo-UTP, and 5-azido-UTP (5), all of which contain crosslinking groups directly on the nucleotide base and function essentially as 0 A probes. Aryl azides are chemically inert until irradiated with long wavelength ultraviolet light, upon which a chemically reactive nitrene is generated. The nitrene can then insert rapidly and relatively nonspecifically into adjacent molecules, resulting in covalent attachment of the azide-tagged molecule to other molecules in the vicinity. This relatively nonspecific insertion reaction makes azides excellent probes for the environment of a molecule, as one specific functional group need not be present on an adjacent molecule to give crosslinking. Using photocrosslinking, proteins that specifically bind to nucleic acids
correspondence should be addressed
Present addresses: *Departnent of Biological Chemistry, University of California, Irvine, CA and +Institute of Molecular Biology, Eugene, OR, USA
University of Oregon,
2074 Nucleic Acids Research, 1993, Vol. 21, No. 9 can be identified, or conformational changes that occur in nucleic acid binding proteins, upon interaction with other molecules, can be detected. We report here the synthesis and characterization of an azide-tagged CTP analog (Fig. 1) and show that it was incorporated enzymatically both at the 3' end and at internal positions in RNA by both E. coli and T7 RNA polymerases. The RNA produced was crosslinked to the RNA polymerases by irradiation of transcription complexes with long wavelength ultraviolet light. The analog was then used to probe for molecular interactions between the E. coli transcription factor NusA and the 3' end of the nascent RNA in a ternary transcription complex. The complex examined is that which is recognized and modified by the bacteriophage X antitermination protein Q, together with NusA, to transform the RNA polymerase to a terminationresistant form.
MATERIALS AND METHODS Buffers and solvents Buffer A: 0.5 M ammonium formate (pH 4.6); buffer B: 3.0 M ammonium formate (pH 4.6); buffer C: 44 mM Tris-HCI (pH 8), 14 mM MgCl2, 0.04 mM EDTA, 20 mM NaCl, 2% (v/v) glycerol, 14 mM ,3-mercaptoethanol, 40 mM acetylated bovine serum albumin; buffer D: 40 mM Tris-HCI (pH 7.6), 15 mM MgCl2, 10 mM ,B-mercaptoethanol, 50 ,ug/ml acetylated bovine serum albumin; buffer E: 7 M urea, 0.1 % (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol; buffer F: 63 mM Tris-HCI, pH 6.8, 50 mM dithiothreitol (DTf), 2% (w/v) SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue. Solvent A: 2 M LiCl; solvent B: 1.0 M LiCl; solvent C: 1.6 M LiCl. DNA templates, enzymes and chemicals The X PR' promoter was provided by Dr Jeff Roberts (Cornell University) on an approximately 830 basepair Bam HI/Hind III fragment containing the entire 6S RNA gene. The fragment was subcloned into the Sma I site of pBluescript® II KS (-) phagemid (Stratagene), and an approximately 850 basepair Bam HI/Hind IH fragment was isolated for transcription. The E. coli ribosomal RNA rrnB P1 promoter was on plasmid pKK5-1 (6), and transcription was carried out on intact plasmid. T7 RNA polymerase and plasmid pLM45 were kindly provided by Dr William McAllister (SUNY). E. coli RNA polymerase (holoenzyme) was purified from MRE 600 cells (Grain Processing Corp.) using published procedures (7,8). NusA was purified as described (9). Calf intestine alkaline phosphatase and snake venom phosphodiesterase were obtained from BoehringerMannheim. Ultrapure nucleotides (Pharmacia) used for transcription were stored in small aliquots at - 80°C. Azidophenacyl bromide, sodium borohydride and CTP were obtained from Sigma Chemical Company. Dimethyl acetamide, dimethyl formamide, sodium sulfide nonahydrate, dimethyl sulfoxide and silver carbonate were from Aldrich Chemical Company. Bromine was from J.T.Baker. Sulfur, ammonium formate, and acetic acid were from Fisher.
Synthesis of 5,5"-dithiobis(cytidine 5'-triphosphate) [bis-5-S-CTP] Bis-5-S-CTP (Fig. 1, IE) was synthesized using a modification of the procedure for bis-5-S-UTP previously described (1). Fresh reagents must be used. CTP (1 g) was suspended in dimethyl
acetamide (70 ml) at 40C. Methylhypobromite (10) was prepared by mixing silver carbonate (28 g) with 75 ml of anhydrous methanol, under nitrogen, in dim light, with a drying tube attached to the flask. The temperature was maintained below - 15°C by placing the flask in a dry ice/ethanol bath. After stirring vigorously for 5 min, 5.6 ml of bromine was added dropwise, and the solution was stirred for 4 hours at or below - 15°C in the dark. The yellow-orange methylhypobromite solution was filtered into an ice-cold flask through Whatman # 1 filter paper on a fritted glass funnel, and the filtrate was immediately added to the CTP/dimethyl acetamide suspension. The reaction was stirred 12 to 24 hours at 40C in the dark. Sodium disulfide pentahydrate was prepared as described (11), 12 g was added to the methoxybromo-CTP solution (Fig. 1, II), and the reaction was stirred 5 hours at 4°C and then 48 hours at room temperature, in the dark. Cold water (500 ml) was added, and the aqueous solution was extracted 3 times with 125 ml watersaturated diethyl ether. The aqueous layer was filtered sequentially through a Whatman # 1 filter followed by a 0.2 ltm filter. The sample was loaded onto a 180 ml AG-IX formate column (BioRad) equilibrated in buffer A and eluted with a 2 L linear gradient of buffer A to buffer B. Bis products eluted as 1 or 2 peaks at approximately 2 M ammonium formate. Different peaks corresponded to bis products containing different combinations of CTP and the CDP and CMP also present in the starting CTP material. The products had absorption maxima at 282-285 nm. Upon reduction with DTT at pH 8.0, a new peak appeared at 332 nm (Fig. 2A) characteristic of the 5-SHpyrimidine (11). All fractions with an absorption maximum of 282-285 nm, and increased absorption at 332 nm after DTT addition, were pooled and lyophilized to remove salt before they were used in the 5-APAS-CTP synthesis. Purity was assessed by HPLC analysis of the pooled fractions on a Beckman Ultrasphere ODS column (4.6 mm x25 cm) using a 30 min linear gradient from 0% to 6% (v/v) acetonitrile in 50 mM triethyl ammonium bicarbonate (TEAB) (pH 8.0) (Fig. 2B). Yields were estimated using the extinction coefficient for 5-SH-uridine of E332=8820 M-'cm-1 at 332 nm, pH 8 (11).
Synthesis and purification of 5-((4-azidophenacyl)thio)cytidine-5'-triphosphate All procedures were carried out in dim light. For reduction of bis-5-S-CTP (M) to 5-SH-CTP (IV), sodium borohydride (10 mg) was added to bis-5-S-CTP (25 Amol), dissolved in 10 ml water. The pH was adjusted to 9.0, and then the sample was stirred on ice for 2-3 hours. The reaction was quenched by adding acetic acid in 10-20 y1 aliquots until the pH was between 4 and 5 and no more gas evolved. The pH was then readjusted to 9.0 with ammonium hydroxide. Azidophenacyl bromide (120 itmol) was dissolved in 3 ml 85% (v/v) dimethyl sulfoxide and added to the reduction reaction. The reaction was stirred overnight at 40C in the dark, and then it was diluted to 150 ml with water and extracted 3 times with water-saturated diethyl ether. The 5-APAS-CTP was purified on a Beckman Ultrrep C18 column, (21.2 mmx 15 cm) using a 30 min gradient of acetonitrile in 50 mM TEAB, pH 7.5. The gradient ran from 8.8% (v/v) to 10% acetonitrile in 5 min, 10% to 14% from 5 to 10 min, and 14% to 15 % from 10 min to 30 min. The 5-APAS-CTP eluted at 12 min and had an absorption maximum at 295 nm. The solvent was removed by lyophilization, and the product was dissolved in dietylpyrocarbonate-treated water. The product concentration
Nucleic Acids Research, 1993, Vol. 21, No. 9 2075 was determined using E293 = 2x 104 M-Icm-I for the azide (12, 13). Product was analyzed by thin-layer chromatography on PEIcellulose F plates developed successively for 4 min in solvent A, 12 min in solvent B, and solvent C until the solvent front reached the top of the plate (Fig. 2C). The plates were not allowed to dry between solvent changes. Product structure was verified by digestion with calf intestine alkaline phosphatase and snake venom phosphodiesterase, as described for 5-APAS-UTP (3). Structure was also verified by nuclear magnetic resonance. The product was lyophilized to dryness and then reconstituted three times in 99.96% D20. The proton NMR was taken in a GN 500-MHz NMR spectrometer.
Transcription and crosslinking reactions Reactions were done in dim light and then split and irradiated or kept in the dark, as described. Preparation of transcription complexes with E. coli RNA polymerase. Ternary transcription complexes (DNA, polymerase, and RNA) containing no analog in the RNA were prepared from two different promoters (Fig. 3, templates 1 and 2), column purified, and the RNA was then elongated in the presence of analog. Transcription complexes containing no analog were prepared from the rrnB P1 promoter, on plasmid pKK5-1 (6), by initiating transcription with 100 nM DNA, 100 nM E.coli RNA polymerase, 100 ,uM ApCpU for 1 min at 30°C in buffer C. The RNA was elongated to G-34 (RNA 34 nucleotides long, terminating in G) by incubation with [a-32P] GTP (3000Ci/mmol), 10 4IM ATP and 10 ,M CTP for 3 min at 30°C.
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Figure 1. Synthesis of 5-APAS-CTP from CTP. 5-APAS-CTP was synthesized by first converting CTP (I) to 5-bromo-6-methoxy-CTP (II) with methyl hypobromite. This was then treated with sodium disulfide to yield 5,5"-dithiobis (cytidine 5'-triphosphate) (Ill, abbreviated bis-5-S-CTP). The bis-5-S-CTP was reduced with sodium borohydride to 5-mercapto-CTP (IV, 5-SH-CTP). The photocrosslinking group was added by alkylation of 5-SH-CTP with pazidophenacyl bromide to give 5-((4-azidophenacyl) thio) cytidine-5'-triphosphate (V, 5-APAS-CTP).
The total reaction volume was 200 ,^l. G-34 complexes were separated from the unincorporated nucleotides by passage through a Sephadex G-50 spun column equilibrated in buffer C. The 5-APAS-CMP was placed uniquely at the 3' end of the RNA by elongation of G-34 RNA with 10 /tM UTP and 200 ltM 5-APAS-CTP for 5 min at 37°C. The analog was placed internally by chasing the G-34 RNA with 10 /M ATP, GTP, UTP and 200 /M 5-APAS-CTP. Control reactions lacking analog or other nucleotides were as described in Figure 4A. From the X PR' promoter (Fig. 3, template 2) a ternary transcription complex containing RNA of 15 nucleotides terminating in UMP (U-15) was made and isolated (Fig. 4B). An initiation complex was formed by incubating 100 Am ApApC, 20 nM DNA (Bam HI/Hind III fragment) and 100 nM E.coli RNA polymerase in buffer C at 30°C for 1 min. The trinucleotide was then elongated to the U-15 RNA by adding 25 tiM ATP, 40 ,tM UTP and 1.5 /tM [a-32P] GTP (3000Ci/mmol) and incubating the reaction at 30°C for 5 min. The transcription complex was separated from free nucleotides by passage through a Sephadex G-50 spun column equilibrated in buffer C. Analog was placed uniquely at the 3' end of the RNA by elongating the U-15 RNA with 200 tiM 5-APAS-CTP for 5 min at 30°C in the presence or absence of 0.8 t4M NusA.
Preparation of transcription complexes with 77RNA polymerase. Transcription complexes were prepared from the T7 RNA polymerase c 10 promoter from plasmid pLM45 (Fig. 3, template 3). Complexes were prepared by incubation of 100 nM DNA, 100 nM T7 RNA polymerase, 100 tiM GpG, 1 tiM [a-32P] GTP (3000 Ci/mmol), 20,tM ATP, and 100 tiM 5-APAS-CTP or 20 tiM CTP (controls) in buffer D for 5 min at 37°C (Fig. 4C). Irradiation and electrophoresis After formation of the desired ternary transcription complex, reactions were split in half, one half for irradiation and the other for the nonirradiated control. Samples were irradiated for 2-4 minutes at room temperature in borosilicate tubes placed 1 to 3 cm from a high intensity 302 nm UV light source (Spectroline model XX-15B, 1800 microwatts/cm2 at 15 cm). Control reactions were kept in the dark during the irradiation, and then DTT was added to all reactions to a final concentration of 60 mM. Samples were then transferred to the respective load buffers for RNA or protein gel analysis. For RNA analysis, samples were added to an equal volume of buffer E, heated for 2 min at 90°C, and run on 40 cmxO.4 mm gels containing 7 M urea and 20 or 25 % polyacrylamide (acrylamide/methylene bisacrylamide = 19: 1), until the bromophenol blue had migrated to the bottom of the gel. For analysis of protein, samples were adjusted to buffer F, heated for 2 min at 90°C, and run on 30 cmxO.7 mm gels containing 7.5% polyacrylamide (acrylamide/methylene bisacrylamide = 37.5:1) and SDS, with a 3.1% stacking gel, or on a 12.5% SDS-polyacrylamide Phast gel (Pharmacia). Protein gels were silver-stained, dried, and exposed to X-ray film at -80°C with an intensifying screen. RNA gels were not dried before exposure to film.
RESULTS AND CONCLUSIONS Synthesis and characterization of 5-APAS-CTP We have synthesized and isolated a new photocrosslinking CTP analog that can be placed either at the 3' end or at internal positions in RNA by both T7 and E. coli RNA polymerases. This
2076 Nucleic Acids Research, 1993, Vol. 21, No. 9
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