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RNA Structure Inhibits the TRAP ( rp RNA-binding AttenuationProtein)-RNA Interaction

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27146

1083-351X

Sandhya Xirasagar, Matthew B. Elliott, Wilmin Bartolini, Paul Gollnick, Philip A. Gottlieb,

Bacterial Genetics and Biotechnology

TRAP (t rp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli. TRAP (t rp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli. trp RNA-binding attenuation protein 7-deazaguanosine. The Bacillus subtilisTRAP1 protein (t rp RNA-bindingattenuation protein) negatively regulates expression of the tryptophan biosynthetic (trp) genes in response to tryptophan (1Babitzke P. Mol. Microbiol. 1997; 26: 1-9Crossref PubMed Scopus (73) Google Scholar, 2Gollnick P. Mol. Microbiol. 1994; 11: 991-997Crossref PubMed Scopus (81) Google Scholar). Upon binding tryptophan, TRAP associates with two specific RNA targets and regulates transcription of thetrpEDCFBA operon (1Babitzke P. Mol. Microbiol. 1997; 26: 1-9Crossref PubMed Scopus (73) Google Scholar, 2Gollnick P. Mol. Microbiol. 1994; 11: 991-997Crossref PubMed Scopus (81) Google Scholar, 3Kuroda M.I. Henner D. Yanofsky C. J. Bacteriol. 1988; 170: 3080-3088Crossref PubMed Google Scholar, 4Babitzke P. Yanofsky C. Proc. Natl. Acad Sci. U. S. A. 1993; 90: 133-137Crossref PubMed Scopus (110) Google Scholar) as well as translation of trpE (5Merino E. Babitzke P. Yanofsky C. J. Bacteriol. 1995; 177: 6362-6370Crossref PubMed Google Scholar) and trpG (6Yang M. Saizieu A. van Loon A.P.G.M. Gollnick P. J. Bacteriol. 1995; 177: 4272-4278Crossref PubMed Google Scholar, 7Du H. Tarpey R. Babitzke P. J. Bacteriol. 1997; 179: 2582-2586Crossref PubMed Google Scholar). The RNA-binding sites for TRAP contain multiple GAG or UAG (rarely AAG) triplet repeats, generally separated by two or three spacer nucleotides (8Babitzke P. Stults J.T. Shire S.J. Yanofsky C. J. Biol. Chem. 1994; 269: 16597-16604Abstract Full Text PDF PubMed Google Scholar, 9Antson A.A. Otridge J. Brzozowski A.M. Dodson E.J. Dodson G.G. Wison K.S. Smith T.M. Yang M. Kurecki T. Gollnick P. Nature. 1995; 374: 693-700Crossref PubMed Scopus (166) Google Scholar). Thetrp leader RNA contains 11 triplet repeats and the binding site in trpG contains 9 repeats. Results from experiments using artificial RNA-binding sites have demonstrated that these trinucleotide repeats are crucial for TRAP binding (10Babitzke P. Bear D.G. Yanofsky C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7916-7920Crossref PubMed Scopus (67) Google Scholar, 11Babitzke P. Yealy J. Campanelli D. J. Bacteriol. 1996; 178: 5159-5163Crossref PubMed Scopus (51) Google Scholar, 12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). These studies also indicate that two nucleotide spacer regions are optimal. Lack of sequence conservation, as well as mutagenesis and footprinting experiments have suggested that the spacer nucleotides do not directly interact with the protein (8Babitzke P. Stults J.T. Shire S.J. Yanofsky C. J. Biol. Chem. 1994; 269: 16597-16604Abstract Full Text PDF PubMed Google Scholar, 9Antson A.A. Otridge J. Brzozowski A.M. Dodson E.J. Dodson G.G. Wison K.S. Smith T.M. Yang M. Kurecki T. Gollnick P. Nature. 1995; 374: 693-700Crossref PubMed Scopus (166) Google Scholar). The crystal structure of TRAP complexed with l-tryptophan reveals that TRAP is an oligomeric protein with 11 identical subunits arranged in a symmetrical ring (9Antson A.A. Otridge J. Brzozowski A.M. Dodson E.J. Dodson G.G. Wison K.S. Smith T.M. Yang M. Kurecki T. Gollnick P. Nature. 1995; 374: 693-700Crossref PubMed Scopus (166) Google Scholar, 13Antson A.A. Brzozowski A. Dodson E. Dauter E. Wilson K. Kurecki T. Otrdige J. Gollnick P. J. Mol. Biol. 1994; 244: 1-5Crossref PubMed Scopus (47) Google Scholar). The secondary structure of TRAP is entirely comprised of β-strands, β-turns, and random coils. Four β-strands from one subunit combine with 3 β-strands from the adjacent subunit, resulting in a novel quaternary structure consisting of 11 7-stranded antiparallel β-sheets. Mutagenesis studies have identified three residues in TRAP, Lys-37, Lys-56, and Arg-58 that when substituted with alanine decrease the affinity of TRAP for trp leader RNA 600–800-fold without altering its ability to bind tryptophan (14Yang M. Chen X.-P. Militello K. Hoffman R. Fernandez B. Baumann C. Gollnick P. J. Mol. Biol. 1997; 270: 696-710Crossref PubMed Scopus (65) Google Scholar). Structural analysis revealed that 11 clusters of this KKR motif, comprised of Lys-37 from one subunit and Lys-56 and Arg-58 from the adjacent subunit, are directly aligned and encircle the TRAP oligomer. This spatial arrangement suggests a model in which the trp leader RNA wraps around TRAP with the (G/U)AG repeats interacting with these 3 amino acid residues (14Yang M. Chen X.-P. Militello K. Hoffman R. Fernandez B. Baumann C. Gollnick P. J. Mol. Biol. 1997; 270: 696-710Crossref PubMed Scopus (65) Google Scholar). This model implies that the RNA would have to be flexible in order to wrap around the protein. Consistent with this hypothesis, we found that the spacer nucleotides in RNA sequences selected for TRAP binding in vitro were predominantly pyrimidines, which stack less well than purines and allow greater flexibility in the RNA (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Arginine residues have been shown to be important in several other protein-RNA interactions (15Tao J. Frankel A.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2723-2726Crossref PubMed Scopus (170) Google Scholar, 16Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (784) Google Scholar). Mutagenic analysis of Arg-58 in TRAP revealed that proteins with substitutions of alanine (R58A), glutamine (R58Q), and lysine (R58K) at this position display a broad range of activity (14Yang M. Chen X.-P. Militello K. Hoffman R. Fernandez B. Baumann C. Gollnick P. J. Mol. Biol. 1997; 270: 696-710Crossref PubMed Scopus (65) Google Scholar). Whereas R58A bound trp leader RNA with greater than 600-fold lower affinity than wild-type TRAP, glutamine substitution was less deleterious and replacement with lysine had no effect on RNA binding. These studies suggested a role for hydrogen bonding interactions between this residue and the RNA in the TRAP-RNA complex. Furthermore, although lysine at this position allows TRAP to function similar to wild-type TRAP with regard to RNA binding and gene regulation, residue 58 is an arginine in TRAP proteins from three different bacilli, suggesting an important role for arginine at this position (17Gollnick P. Ishino S. Kuroda M.I. Henner D.J. Yanofsky C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8726-8730Crossref PubMed Scopus (66) Google Scholar, 18Hoffman R.J. Gollnick P. J. Bacteriol. 1995; 177: 839-842Crossref PubMed Google Scholar). 2X. Chen, C. Baumann, and P. Gollnick, unpublished results. These studies led us to further examine the role of Arg-58 in the TRAP-RNA interaction. To test the role of this residue in specificity determination, we compared the sequences of RNAs that bind to wild-type TRAP to those that bind R58K TRAP using SELEX (19Tuerk C. Gold L. Science. 1990; 249: 505-510Crossref PubMed Scopus (8055) Google Scholar). RNAs selectedin vitro for binding to R58K TRAP showed a bias for UAG over GAG repeats, whereas RNAs selected with wild-type TRAP contained more GAG repeats. These results suggested that Arg-58 may interact with the first nucleotide of each triplet repeat in the RNA, possibly including an interaction with the nitrogen at position 7 (N-7) of guanosine. However, RNAs containing the analogue 7-deazaguanosine (C7G), in which the N-7 is replaced by carbon, showed an unexpected enhanced affinity for TRAP, especially in the case of R58K TRAP. This effect was independent of the position of the analogue in the (G/U)AG repeat but was only observed with RNAs containing CC spacers. Effects of temperature and Mg2+ on the binding of RNAs containing 11 GAG or UAG repeats with UU or CC spacers, as well as thermal denaturation studies of these RNAs demonstrated that ((G/U)AGCC)11 RNAs form stable structures that inhibit binding to TRAP. The exact nature of this structure is not yet known, however, we present evidence that it involves interactions other than Watson-Crick base pairing. Furthermore, although our studies did not support a direct interaction between Arg-58 and the first nucleotide in each triplet repeat, they did indicate a role for Arg-58 in binding to structured RNAs. The nucleoside 7-deazaguanosine was synthesized as described previously (20Ramasamy K. Imamura N. Robins R.K. Revankar G.R. Tetrahedron Lett. 1987; 28: 5107-5110Crossref Scopus (46) Google Scholar). Conversion to the triphosphate was achieved by reacting 50 mg of the nucleoside analogue with 34 μl of POCl3 in 100 μl of trimethyl phosphate. Progress of the reaction was followed by silica gel TLC usingn-Pr-OH:NH4OH:H2O (6:3:1). After 1 h, 2 ml of 1 m pyrophosphate tributylammonium salt (Sigma) in freshly distilled dimethylformamide was added. After 10 min, the reaction solution was cooled to 4 °C and quenched by adding 2 ml of triethylamine and 4 ml of H2O and then stored at −20 °C. Purification was achieved by anion exchange chromatography with DEAE-cellulose (22 × 1.5 cm; Whatman DE-52) as described previously (21Wiecsorek A. Dinter-Gottlieb G. Gottlieb P.A. Bioorg. Med. Chem. Lett. 1994; 4: 987-994Crossref Scopus (5) Google Scholar). The yield of the triphosphate was 1.65 mg. UV (pH 7.0) λmax 218, 260 (shoulder peak 280–290), λmin 238. 1H NMR (D2O; 400 MHz) δ = 3.95–4.09 (2 H, m) 4.15 (1 H, m) 4.36 (1 H, m) 4.55 (1 H, m) 5.89 (1 H, d, J = 7.13) 6.42 (1 H, d, J= 3.76) 7.03 (1 H, d, J = 3.83); 31P NMR (D2O; 161 MHz) δ = −9.94 (d), −10.77 (d), −22.55 (t). All plasmids were propagated in Escherichia coli JM107. Plasmid pTZ18UAGCC has 11 repeats of the sequence 5′-TAGCC-3′ cloned downstream of the T7 promoter in pTZ18U (USB). The insert was created by annealing oligonucleotides B (5′-TCTAAGCTTGGCTAGGCTAG-3′) and C (5′-AGAGAATTC(TAGCC)11AAGCT TAGA-3′). The 3′ ends were extended using the Klenow fragment of DNA polymerase I and the resulting duplex DNA was digested with EcoRI and HindIII and ligated into similarly digested pTZ18U. With the exception of the sequence of the oligonucleotides that were used to make the duplex DNA, plasmids pTZ18UAGUU and pTZ18GAGUU were made by a similar approach. Oligonucleotides D (5′-AGGGAATTC(TAGTT)10TAGAAGCTTGGTCGTGACTGGGAAAAC-3′) and −40 (5′-GTTTTCCCAGTCACGAC-3′) were used to construct plasmid pTZ18UAGUU with 10 repeats of the sequence 5′-TAGTT-3′ followed by TAGA. Plasmid pTZ18GAGUU, with the sequence 5′-GAGTT-3′ repeated 10 times followed by GAGA was constructed using oligonucleotides E (5′-AGGGAATTC(GAGTT)10GAGAAGCTTGGTCGTGACTGGGAAAAC-3′) and −40 (see above). Plasmid pSP64GAGCC contains 11 repeats of the sequence 5′-GAGCC-3′ cloned downstream of the T7 promoter. To construct this plasmid, oligos T7 (5′-AGGAATTCAATTATAATACGACTCACTATA-3′) and F (5′-GAGGGCCC (GGCTC)11TATAGTGAGTCG-3′) were used to make the duplex DNA, which was digested with EcoRI and Bsp120I and ligated into similarly digested pSP64/p36T (22Ziehler W.A. Engelke D.R. BioTechniques. 1996; 20: 622-624PubMed Google Scholar). In vitrosynthesis of 32P-labeled RNA using T7 RNA polymerase has been described previously (23Baumann C. Otridge J. Gollnick P. J. Biol. Chem. 1996; 271: 12269-12274Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For 7-deazaguanosine (C7G) substituted RNAs, GTP was substituted with C7GTP (2 mm), and GMP (2.5 mm) was included to allow initiation of transcription. Templates for transcription were generated by linearizing the appropriate plasmids with HindIII. For the sake of convenience, we will designate the RNA with 10 GAGUU repeats followed by GAGA as (GAGUU)11, that with 10 UAGUU repeats followed by UAGA as (UAGUU)11, that with 11 UAGCC repeats, as (UAGCC)11, and that with 11 GAGCC repeats, as (GAGCC)11. All of the transcripts, except (GAGCC)11, contain the sequence GGGAAUUC at their 5′ ends. To synthesize unlabeled RNAs, linearized templates were incubated in T7 transcription buffer (Life Technologies, Inc.) with 10 mmdithiothreitol, 100 mm MgCl2, 4 mmGTP, ATP, CTP, and UTP and T7 RNA polymerase at 37 °C for 2 h. Transcription of (UAGUU)11 was performed at room temperature because the reaction was most efficient at this temperature. All RNAs were gel-purified and quantitated as described previously (23Baumann C. Otridge J. Gollnick P. J. Biol. Chem. 1996; 271: 12269-12274Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Wild-type and R58K TRAP were purified as described previously (13Antson A.A. Brzozowski A. Dodson E. Dauter E. Wilson K. Kurecki T. Otrdige J. Gollnick P. J. Mol. Biol. 1994; 244: 1-5Crossref PubMed Scopus (47) Google Scholar, 24Otridge J. Gollnick P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 128-132Crossref PubMed Scopus (86) Google Scholar). Quantitative filter binding assays were performed in filter binding buffer (250 mm potassium glutamate, 16 mm HEPES pH 8.0, and 4 mmMgCl2) as described previously (23Baumann C. Otridge J. Gollnick P. J. Biol. Chem. 1996; 271: 12269-12274Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), except that the reactions were incubated for 1 h instead of 15 min, because this time was required for binding to reach equilibrium with some of the RNAs studied. The data was analyzed using a nonlinear least squares fitting algorithm (Psi Plot, Poly Software International, Sandy UT). Data are the average of at least two individual experiments. SELEX experiments with R58K TRAP were performed as described previously for wild-type TRAP (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). An oligonucleotide pool with 25 random positions, which can accommodate five trinucleotide repeats each separated by two nucleotide spacers was used. This pool size (425) was well represented by 2 nmol of synthetic oligonucleotide. RNAs that bound to TRAP were selected by filter binding and were eluted from the nitrocellulose filters as described previously (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Eight rounds of SELEX were performed and selected RNAs were reverse transcribed, the cDNAs cloned in to pUC118 and individual clones sequenced using Sequenase (Amersham). Temperature-dependent UV absorption data of RNAs (5 μm in 250 mm KCl and 16 mm HEPES pH 8.0) were collected with a Beckman DU-640 spectrophotometer using a circulating water bath with a heating rates of either 1 °C or 0.25 °C per min. Both heating rates gave identical results. Data were normalized to the absorbance at 16 °C. CD spectra were obtained with a JASCO model J-500C spectropolarimeter using 1-mm path length cuvettes. CD spectra of RNAs (2.5 μm) were recorded between 25 and 80 °C in 50 mm potassium phosphate, pH 7.4. For studies of TRAP binding, 2.5 μm RNA was incubated with 5 μm wild-type TRAP and 50 μml-tryptophan at 55 °C for 1 h. Neither TRAP nor tryptophan show significant dichroism in the range in which the data were recorded (230–300 nm) and therefore make no contribution to the spectra. Previous studies identified Arg-58 as one of three residues important for RNA binding to TRAP (14Yang M. Chen X.-P. Militello K. Hoffman R. Fernandez B. Baumann C. Gollnick P. J. Mol. Biol. 1997; 270: 696-710Crossref PubMed Scopus (65) Google Scholar). To further investigate the role of Arg-58 in the specificity of this interaction, we used SELEX to select from a pool of 1.2 × 1015RNAs containing 25 random positions, those RNAs that bound R58K TRAP with highest affinity (19Tuerk C. Gold L. Science. 1990; 249: 505-510Crossref PubMed Scopus (8055) Google Scholar). We then compared the sequence requirements for RNA binding to R58K TRAP to those for binding to wild-type. RNAs selected for binding to R58K TRAP were similar to those found previously with wild-type TRAP (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), in that they all contained multiple GAG and UAG repeats (Fig. 1 and Table I).Table IAnalysis of RNA sequences that bind to wild-type and R58K TRAPRepeats1-a% GAG and % UAG refers to their percentage among the triplet repeats in all of the selected RNAs. Repeats where the first position came from nonrandom flanking sequence were not considered in this calculation.Spacers1-bSpacers are defined as nucleotides between two adjacent (G/U)AG repeats.% GAG% UAG%2 Nucleotides% Pyrimidines% U% C% A% GTRAP WT1-cData for wild-type TRAP has been reported previously (12) and is included here for comparison with the mutants. One CAG repeat was also included in this calculation.64358988741411.30.7R58K1-d45 cDNA clones contained a total of 158 repeats consisting of 30 GAG and 128 UAG repeats with 113 spacers.1981909078129.50.5RNAs were selected using SELEX from a pool containing 25 positions with random nucleotides.1-a % GAG and % UAG refers to their percentage among the triplet repeats in all of the selected RNAs. Repeats where the first position came from nonrandom flanking sequence were not considered in this calculation.1-b Spacers are defined as nucleotides between two adjacent (G/U)AG repeats.1-c Data for wild-type TRAP has been reported previously (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and is included here for comparison with the mutants. One CAG repeat was also included in this calculation.1-d 45 cDNA clones contained a total of 158 repeats consisting of 30 GAG and 128 UAG repeats with 113 spacers. Open table in a new tab RNAs were selected using SELEX from a pool containing 25 positions with random nucleotides. The spacers separating the (G/U)AG repeats were also similar to those obtained previously with wild-type TRAP (12Baumann C. Xirasagar S. Gollnick P. J. Biol. Chem. 1997; 272: 19863-19869Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), being predominately 2 nucleotides (90%), with 1 (8%) and 3 nucleotides (2%) spacers rarely observed (Table I). The composition of the spacers was also similar to that observed previously with wild-type TRAP, being predominately (>90%) pyrimidines with greater than 75% uridines. We suggested previously that this bias for pyrimidines reflects their lower stacking ability (as compared with purines) allowing greater flexibility for the RNA to wrap around the protein. However, this hypothesis does not explain the preponderance of U's in the spacers since both C's and U's are predicted to stack similarly (25Broyde S. Hingerty B. Nucleic acids Res. 1978; 5: 2729-2741Crossref PubMed Scopus (8) Google Scholar) and thus would be expected to be present with comparable frequencies if flexibility is the only consideration. Furthermore, few if any C's occurred as CC dinucleotide spacers. In RNAs obtained with wild-type TRAP, 75% of C's in the spacers were observed as CU, 7% as UC, and 6% each as AC, CA, or CC dinucleotides. With R58K TRAP, 88% of the C's occurred as CU spacers, and 4% each as UC, CA, or single nucleotide C spacers. These results suggest that C's in spacers are inhibitory to TRAP binding, particularly as CC spacers. These findings are consistent with previous studies of CU spacers in RNAs with 6 GAG repeats (11Babitzke P. Yealy J. Campanelli D. J. Bacteriol. 1996; 178: 5159-5163Crossref PubMed Scopus (51) Google Scholar). The only significant difference we observed between RNAs that bound R58K TRAP as compared with wild-type TRAP was the ratio of GAG to UAG repeats in the RNAs. While RNAs that bound wild-type TRAP contained more GAGs, RNAs obtained with R58K TRAP contained predominantly UAG repeats (Table I). These results indicate that amino acid substitution at position 58 in TRAP can alter the preference for the base in the first position of the trinucleotide repeat, suggesting that Arg-58 may interact directly with the first G or U in the triplet. One mechanism by which Arg-58 could interact with the first guanosine of a GAG repeat would be by forming two hydrogen bonds with the O-6 and N-7 positions of the base. Such bidentate interactions between arginines and guanosines have been demonstrated to be important in other protein-nucleic acid complexes (15Tao J. Frankel A.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2723-2726Crossref PubMed Scopus (170) Google Scholar, 16Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (784) Google Scholar). This model makes several predictions about the interaction of TRAP with RNA. First, wild-type TRAP should have a much higher affinity for GAG- than for UAG-containing RNAs because arginine can form two hydrogen bonds with guanosine but only one with uracil. This would result in a total of 11 additional hydrogen bonds in a complex between wild-type TRAP and an RNA with 11 GAG repeats as compared with one with 11 UAG repeats (ΔΔG o = 11–22 kcal/mol) (26Lesser D.R. Kurpiewski M.R. Jen-Jacobson L. Science. 1990; 250: 776-785Crossref PubMed Scopus (304) Google Scholar, 27Mazzarelli J.M. Rajur S.B. Ladarola P.L. McLaughlin L.W. Biochem. 1992; 31: 5925-5936Crossref PubMed Scopus (42) Google Scholar, 28Zhang X. Gottlieb P.A. Biochem. 1993; 32: 11374-11385Crossref PubMed Scopus (17) Google Scholar). Conversely, R58K TRAP should have similar affinities for both GAG and UAG repeat containing RNAs since lysine can only form one hydrogen bond with either guanosine or uridine. We therefore examined the affinities of wild-type and R58K TRAP for RNAs with 11 GAG repeats ((GAGUU)11) or 11 UAG repeats ((UAGUU)11) (Table II). In contrast to our predictions, at 37 °C in the presence of Mg2+, wild-type TRAP bound (GAGUU)11 and (UAGUU)11 with equal affinities (K d = 0.3 nm). In addition, both wild-type and R58K TRAP bound similarly to both RNAs, although R58K displayed a slightly lower affinity for (GAGUU)11 (K d = 0.8 nm) than for (UAGUU)11 (K d = 0.3 nm). Moreover, at higher temperature (55 °C), both R58K and wild-type TRAP behaved identically when binding to these RNAs (Table II). The results of these binding studies are consistent with our SELEX data, however, they do not support the model of bidentate hydrogen bonding between Arg-58 and guanosine in the first position of the triplet repeats.Table IIEffect of Mg2+ and temperature on RNA binding to wild-type and mutant TRAP(UAGUU)11 K d(nm)2-aApparent K d values (as determined by filter binding) represent the averages of at least two independent experiments; standard deviations were less than 10% of the mean.(GAGUU)11 K d (nm)(UAGCC)11 K d (nm)(GAGCC)11 K d (nm)37 °C37 °C55 °C37 °C37 °C55 °C37 °C37 °C55 °C37 °C37 °C55 °CMg2+2-b+ indicates the presence of 4 mm Mg2+.−++−++−++−++TRAP WT0.30.31ND2-cND, not determined.0.30.32020056609R58K0.30.31ND0.80.3300NB2-dNo binding detected up to 3 μm TRAP.60066672-a Apparent K d values (as determined by filter binding) represent the averages of at least two independent experiments; standard deviations were less than 10% of the mean.2-b + indicates the presence of 4 mm Mg2+.2-c ND, not determined.2-d No binding detected up to 3 μm TRAP. Open table in a new tab Although the binding data did not support the model for two hydrogen bonds between the first guanosine of a GAG repeat and Arg-58, they did not rule out a role for N-7 of guanosines in interacting with TRAP. We tested whether the N-7 position of guanosines are involved in binding to TRAP using RNAs substituted with 7-deazaguanosine (C7G), an analogue of guanosine, in which the nitrogen at position 7 is replaced by carbon (Fig. 2). If the N-7 of guanosine interacts with TRAP via hydrogen bonding, substitution with C7G will eliminate this interaction and therefore lower the affinity of the substituted RNA for TRAP. We were particularly interested in the first position of the triplet repeat, since our studies suggested a possible interaction between Arg-58 and guanosine in this position. However, since it is not possible to specifically replace only the first guanosine of each GAG repeat by in vitro transcription, we first tested the effect of C7G in the third position of each UAG repeat in the RNA (UAGCC)11. We then compared this effect with that of substituting the analogue at both the first and the third positions in each GAG repeat, using RNAs (GAGCC)11and (GAGUU)11. Surprisingly, we found that introduction of C7G in (UAC7GCC)11 did not reduce the affinity of this RNA for either wild-type or R58K TRAP but instead resulted in increased affinity for both proteins as compared with the unsubstituted RNA (Table III). With wild-type TRAP we observed a 30-fold effect, and R58K TRAP bound (UAC7GCC)11 with an apparentK d of 7 nm but showed no detectable binding to the unsubstituted (UAGCC)11 (Table III). Introduction of C7G in both positions of each triplet repeat in (C7GAC7GCC)11 also increased the affinity of this RNA (8–9-fold) relative to the unsubstituted RNA for both wild-type and R58K TRAP (Table III). In contrast to observations with RNAs containing CC spacers,C7G substitution in (C7GAC7GUU)11 had little effect on the affinity of this RNA for wild-type or R58K TRAP (Table III). The observation that incorporation of C7G into either GAG- or UAG-containing RNAs never reduced the affinity of these RNAs for TRAP, and in several cases significantly increased their affinities, suggests that the N-7 of guanosine at neither the first nor the third position of the triplet repeats interacts directly with the protein.Table IIIEffect of 7-deazaguanosine (C7G) on RNA binding to wild-type and mutant TRAP(UAGCC)11 K d3-aApparent K d values were determined by filter binding at 37 °C in the presence of 4 mmMg2+. Values are the averages of at least two independent experiments with standard deviations being less than 10% of the mean.(UAC7GCC)11 K d(GAGCC)11 K d(C7GAC7GCC)11 K d(GAGUU)11 K d(C7GAC7GUU)11 K dnmTRAP WT20076070.30.2R58KNB3-bNo binding detected up to 3 μm TRAP.76670.80.23-a Apparent K d values were determined by filter binding at 37 °C in the presence of 4 mmMg2+. Values are the averages of at least two independent experiments with standard deviations being less than 10% of the mean.3-b No binding detected up to 3 μm TRAP. Open table in a new tab The C7G-induced enhancement in affinity for TRAP is independent of the position of substitution in the (G/U)AG repeats, since it occurred with (UAGCC)11 and (GAGCC)11but not with (GAGUU)11, again suggesting that this effect was not due to changing direct inte