Identification of the RNA Binding Domain of T4 RegA Protein by Structure-based Mutagenesis
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32265
ISSN1083-351X
AutoresJohnthan Gordon, Tapas K. Sengupta, Christine A. Phillips, Shawn M. O'Malley, Kenneth R. Williams, Eleanor K. Spicer,
Tópico(s)RNA modifications and cancer
ResumoThe T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H., Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170–1173). Domain I of the RegA protein contains a four-stranded β-sheet and two α-helices. Domain II contains a four-stranded β-sheet and an unusual 3/10 helix. Since β-sheet residues play a role in a number of protein-RNA interactions, one or both of the β-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both β-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of β-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an α-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties. The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H., Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170–1173). Domain I of the RegA protein contains a four-stranded β-sheet and two α-helices. Domain II contains a four-stranded β-sheet and an unusual 3/10 helix. Since β-sheet residues play a role in a number of protein-RNA interactions, one or both of the β-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both β-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of β-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an α-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties. The bacteriophage T4 RegA protein is a unique translational repressor in that it is able to regulate the expression of 15–30 T4 genes, including its own. Previous studies have demonstrated that RegA protein binds to the translation initiation region of target mRNAs (1Winter R.B. Morrissey L. Gauss P. Gold L. Hsu T. Karam J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7822-7826Crossref PubMed Scopus (61) Google Scholar, 2Webster K.W. Adari H.Y. Spicer E.K. Nucleic Acids Res. 1989; 17: 10047-10068Crossref PubMed Scopus (15) Google Scholar) and inhibits the formation of ribosome-mRNA initiation complexes (1Winter R.B. Morrissey L. Gauss P. Gold L. Hsu T. Karam J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7822-7826Crossref PubMed Scopus (61) Google Scholar, 3Unnithan S. Green L. Morrissey L. Binkley J. Singer B. Karam J. Gold L. Nucleic Acids Res. 1990; 18: 7083-7092Crossref PubMed Scopus (18) Google Scholar). Although the RegA recognition element (RE) 1The abbreviations used are:RErecognition elementWTwild type has been studied in detail in two genes, gene 44 (2Webster K.W. Adari H.Y. Spicer E.K. Nucleic Acids Res. 1989; 17: 10047-10068Crossref PubMed Scopus (15) Google Scholar, 4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar) and the rIIB gene (1Winter R.B. Morrissey L. Gauss P. Gold L. Hsu T. Karam J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7822-7826Crossref PubMed Scopus (61) Google Scholar, 5Karam J. Gold L. Singer B.S. Dawson M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4669-4673Crossref PubMed Scopus (34) Google Scholar), and has been mapped by RNase protection assays in three other mRNAs (3Unnithan S. Green L. Morrissey L. Binkley J. Singer B. Karam J. Gold L. Nucleic Acids Res. 1990; 18: 7083-7092Crossref PubMed Scopus (18) Google Scholar), it is not completely clear what common RNA features allow RegA to recognize its diverse targets. recognition element wild type The solution of the crystal structure of RegA protein by Kang and co-workers (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar) has allowed for inspection of potential RNA binding domains and for comparison of RegA to the structures of other known RNA-binding proteins. Although RegA protein exists as a dimer in crystals (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar) and in solution at moderate concentrations (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar), it binds to a 16-mer RNA corresponding to the gene 44 RE as a monomer (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar). Within the RegA monomer, shown in Fig. 1, there are two structural domains. Domain I consists of a four-stranded β-sheet (β6, β1, β5, and β4) and two α-helices (helix A and helix C). Domain II consists of a second four-stranded β-sheet (β2, β3, β9, and β8) adjacent to an unusual 3/10 helix (helix D). Although photocross-linking (8Webster K.R. Keill S. Konigsberg W. Williams K.R. Spicer E.K. J. Biol. Chem. 1992; 267: 26097-26103Abstract Full Text PDF PubMed Google Scholar), partial proteolysis (9O'Malley S.M. Sattar A.K.M. Williams K.R. Spicer E.K. J. Biol. Chem. 1995; 270: 5107-5114Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), and truncation studies (9O'Malley S.M. Sattar A.K.M. Williams K.R. Spicer E.K. J. Biol. Chem. 1995; 270: 5107-5114Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) have been performed on RegA protein, a definitive localization of the RNA binding site on RegA has not been achieved. In considering where RNA might bind on RegA protein, Kang et al. (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar) noted that β-sheets often are important contributors to RNA recognition. For example, a β-pleated sheet in glutaminyl-tRNA synthetase plays a role in binding the anti-codon region of tRNAGln (10Rould M.A. Perona J.J. Söll D. Steitz T.A. Science. 1989; 246: 1135-1142Crossref PubMed Scopus (804) Google Scholar), and residues in the MS2 coat protein that participate in sequence-specific RNA binding lie on β-strands within an extended 10-stranded β-sheet in the protein dimer (11Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (322) Google Scholar). A β-sheet also plays a critical role in the binding of U1 RNA to the small nuclear ribonucleoprotein U1A protein (12Oubridge C. Ito N. Evans P.R. Teo C-H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (784) Google Scholar). U1A binds to stem-loop II of U1 RNA through interactions between loop nucleotides and residues on the central two β-strands of a four-stranded β-sheet. The central β-strands contain the two highly conserved RNP consensus sequences (RNP-1 and RNP-2) found in over 100 RNA-binding proteins (13Birney E. Kumar S. Krainer A.R. Nucleic Acids Res. 1993; 21: 5803-5816Crossref PubMed Scopus (589) Google Scholar, 14Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1731) Google Scholar) (see Fig. 1). As noted by Kang and co-workers (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar), two strands of the four-stranded β-sheet in domain I of RegA protein exhibit sequence similarities to RNP-1 and RNP-2 (including aromatic residues on β5), suggesting that this region may participate in RNA binding. Further, U1A protein has basic residues in two loops at the base of the β-sheet, referred to as “jaws,” which interact with the backbone of the U1 RNA hairpin. RegA protein also has two pairs of basic residues (Lys7 and Lys8; Lys41 and Lys42) in loops at the base of the β-sheet (see Fig. 1), which could be envisioned to function in binding RNA. Although domain II of RegA protein does not exhibit structural similarity with any known RNA binding protein, there is experimental evidence suggesting that domain II residues contribute significantly to RNA binding. For example, the site of photocross-linking of RegA protein to nucleic acid was found to be Phe106 (8Webster K.R. Keill S. Konigsberg W. Williams K.R. Spicer E.K. J. Biol. Chem. 1992; 267: 26097-26103Abstract Full Text PDF PubMed Google Scholar), which lies within domain II. Also, removal of 13 or 17 residues from the C terminus of RegA (located in domain II) resulted in 100–1000-fold reductions in RNA binding affinity (9O'Malley S.M. Sattar A.K.M. Williams K.R. Spicer E.K. J. Biol. Chem. 1995; 270: 5107-5114Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Finally, partial proteolysis of RegA protein, which leads to cleavage at three sites in the C-terminal domain, is reduced by RNA binding (9O'Malley S.M. Sattar A.K.M. Williams K.R. Spicer E.K. J. Biol. Chem. 1995; 270: 5107-5114Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). In addition to the above experimental observations and the observed similarities of RegA protein with other RNA-binding proteins, the fact that RegA protein is small (14.6 kDa) and binds to a relatively large binding site size on RNA (≥12 nucleotides) (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar) suggests that residues involved in RNA binding may span both domains of the protein. In fact, Kang et al. (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar) have suggested that the two β-sheet regions, which are 25 Å apart, may both participate in RNA binding. To test this possibility, site-specific mutagenesis of surface residues in both domains I and II of RegA protein was performed. Basic and aromatic residues were particularly targeted, since they have been found to play important roles in a number of protein-RNA complexes (11Valegard K. Murray J.B. Stockley P.G. Stonehouse N.J. Liljas L. Nature. 1994; 371: 623-626Crossref PubMed Scopus (322) Google Scholar, 15Shamoo Y Krueger U. Rice L.M. Williams K.R. Steitz T.A. Nat. Struct. Biol. 1997; 4: 215-222Crossref PubMed Scopus (118) Google Scholar). Ten residues that lie within the central β-strands and β-loop regions of domain I were mutated, and the resulting proteins were assayed for RNA binding in vitro. In addition, residues within β-sheet A of domain II were mutated to test for their contribution to RNA binding. During the course of these studies, molecular models of RegA protein were generated to examine surface regions for potential RNA binding pockets and to determine whether residues to be mutated are located at the surface or in the core of the folded protein. These models revealed the presence of a pocket and a deep cleft at opposite sides of the interface between domains I and II. Subsequent mutagenesis was performed to test the potential role of these two regions in RNA binding. Taken together, the results of these mutagenesis and modeling studies have ruled out the participation of a number of proposed functional residues, revealing instead an unexpected site for RNA binding on RegA protein. Oligodeoxyribonucleotides were synthesized on an Expedite (model 8909) nucleic acid synthesizer by the Medical University of South Carolina Oligosynthesis Facility. Oligoribonucleotides were synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University) and were deprotected as described previously (16Webster K.W. Shamoo Y. Konigsberg W.H. Spicer E.K. BioTechniques. 1991; 11: 658-661PubMed Google Scholar) and then purified by perfusion chromatography using PorosTM HQ and R1 columns (PerSeptive Biosciences), essentially as described (16Webster K.W. Shamoo Y. Konigsberg W.H. Spicer E.K. BioTechniques. 1991; 11: 658-661PubMed Google Scholar). Poly(U) and poly(U)-agarose were purchased from Amersham Pharmacia Biotech. Escherichia coli AR120 (λcI+, N+) was obtained from A. Shatzman (Smith, Klein and French); construction of pAS1regA was described previously (2Webster K.W. Adari H.Y. Spicer E.K. Nucleic Acids Res. 1989; 17: 10047-10068Crossref PubMed Scopus (15) Google Scholar). Site-directed mutagenesis of residues Lys7, Lys8, Lys16, Lys41, Lys42, Tyr45, and Tyr46 was carried out by annealing mutagenic oligonucleotides (33–42 nucleotides in length) to a double-stranded plasmid carrying the wild type (WT) regA gene (pAS1regA) (2Webster K.W. Adari H.Y. Spicer E.K. Nucleic Acids Res. 1989; 17: 10047-10068Crossref PubMed Scopus (15) Google Scholar) as described previously (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar). The remainder of the mutations were introduced by the Quik-ChangeTMSite-Directed Mutagenesis protocol (Stratagene). Pfu DNA polymerase was used in a 16-cycle thermal cycling reaction to extend and incorporate the appropriate mutagenic primers, which resulted in nicked circular strands. The methylated, nonmutated parental DNA was then digested with DpnI, and the DNA was transformed intoE. coli AR120 cells. Plasmids were purified from overnight LB/amp cultures (25 ml) using the Qiagen miniprep system. Mutations were then confirmed by DNA sequence analysis, using an ABI 377 automated DNA sequencer, by the Medical University of South Carolina Biotechnology Resource Laboratory. To facilitate purification of selected mutant RegA proteins, the RegA expression vector was modified to code for a fusion protein containing four His residues at the COOH terminus of RegA protein. An oligonucleotide containing four CAT codons was inserted into the pAS1regA vector between the terminal codon (AAT) and the stop codon (TAA) of regA, using the Quik-ChangeTM protocol. Insertion of the 12-nucleotide sequence was confirmed by DNA sequencing. Mutations K14A, T18A, R21A, and W81A were then introduced into the regA-His4 vector. Wild type (WT), K7L, K41L, and K42L RegA proteins were purified from AR120 cells containing WT or mutant pAS1regA plasmids following induction of transcription from the phage λ PL promoter by nalidixic acid treatment, as described previously (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar, 17Adari H.Y. Rose K. Williams K.W. Konigsberg W.H. Lin T.-C. Spicer E.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1901-1905Crossref PubMed Scopus (12) Google Scholar). Protein concentrations for fluorescence analysis were determined by duplicate amino acid analyses, performed by the W. M. Keck Foundation Biotechnology Laboratory (Yale University). The expected error in the resulting concentrations is less than ±10%. RegA proteins carrying a C-terminal His4 fusion were purified by perfusion chromatography using a PorosTM MC column charged with Ni2+ on a Biocad SPRINTTMchromatography system (PerSeptive Biosciences). Induced cell extracts were centrifuged at 100,000 × g for 1 h, and the supernatant was dialyzed into 20 mm phosphate (pH 7.5), 200 mm NaCl, 10 mm imidazole (buffer A) overnight. The supernatant was applied to a 1.7-ml MC column equilibrated in buffer A. The column was washed with 5 column volumes of buffer A and then eluted with a gradient of 10–200 mm imidazole in buffer A. Column fractions were analyzed by SDS-polyacrylamide gel electrophoresis and then pooled and concentrated by centrifugation through Centriprep 10TM (Amicon, Inc.) filtration units. Fluorescence quenching assays were performed at 25 °C using an SLM model 8000C spectrofluorometer (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar). Reverse titrations (addition of poly- or oligoribonucleotide lattice to protein ligand) were performed in 2-ml stirred cuvettes at protein concentrations of 0.1–1.0 μm in buffer C (10 mm HEPES, pH 7.2, 5 mm MgCl2, 1 mm EDTA, and 1 mm β-mercaptoethanol) (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar) plus 150 mm NaCl (for gene 44 RE RNA) or 20 mm NaCl (for poly(U)). Data were acquired at an excitation wavelength of 282 nm and an emission wavelength of 347 nm. The effects of photobleaching during titrations were corrected for by monitoring RegA protein fluorescence in a control cuvette. The average photobleaching control was 7.3%. Correction for absorption of incident light by oligonucleotides was made by performing a parallel titration ofN-acetyl-l-tryptophanamide (Sigma) with nucleic acid. The effect of salt on RNA affinity was determined by “salt-back” titration (18McSwiggen J.A. Bear D.G. Von Hippel P.H. J. Mol. Biol. 1988; 199: 609-622Crossref PubMed Scopus (85) Google Scholar), in which NaCl was added stepwise to RegA protein-poly(U) complexes following reverse titrations. The apparent association constant (Kapp) for poly(U) was calculated from the equivalence point of the titration (i.e.at the addition of an equimolar amount of RNA), assuming a binding site size of n = 9 nucleotides (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar). A single binding site was assumed for RegA protein binding to gene 44 RE (16-mer) RNA (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar,7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar). Cell supernatants were prepared by the method of Johnson and Hecht (19Johnson B.H. Hecht M.H. Bio/Technology. 1994; 12: 114-117Google Scholar), with slight modifications. Briefly, transformed E. coliAR120/pAS1regA cells were grown at 37 °C to anA590 nm of 0.8–0.9, and then WT or mutantRegA expression was induced for ∼15 h by the addition of nalidixic acid (80 μg/ml). After centrifugation of a 100-ml culture, cell pellets were frozen in an ethanol/dry ice bath for 2 min, followed by thawing in an ice water bath for 8 min, repeated for a total of four cycles. The pellet was resuspended in 800 μl of 20 mmTris-HCl, pH 8.0, 100 mm NaCl, and 1 mm EDTA and incubated in an ice water bath for 30 min. The suspension was centrifuged in a microcentrifuge for 3 min; the supernatant was recovered, and the pellet was resuspended in 1 ml of 1× TE (10 mm Tris, 1 mm EDTA, pH 7.4). Cell supernatants were stored in 80-μl aliquots at −20 °C. Evaluation of the protein content of both the supernatant and the pellet resuspension was carried out by SDS-polyacrylamide gel electrophoresis. Some mutant RegA proteins were not completely soluble when produced at 37 °C, and in those cases, induction was repeated at 25 °C. In each case, solubility was increased sufficiently to allow gel shift assays to be performed. RegA protein concentrations in cell lysates were determined by quantitation of protein fluorescence in gels stained with SYPRO Orange (Molecular Probes, Inc., Eugene, OR) compared with a standard curve of known concentrations of purified RegA protein, using a Molecular Dynamics Storm Imager. Protein concentrations had an error range of 1–16% (average = 7%). Purified gene 44 RE RNA (5′-GAAUGAGGAAAUUAUG-3′) was 5′-32P-end-labeled by treatment with T4 polynucleotide kinase and [γ-32P]ATP (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 5.86Google Scholar). Increasing volumes of induced cell supernatants were incubated with a constant amount of 32P-labeled gene 44 RE RNA (10 nm) to generate titration curves. Binding was carried out in 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, and 1 mm EDTA at 4 °C for 15 min. Binding reactions were performed in a 20-μl final volume with final concentrations of 2.5–40 nm RegA protein. A freshly thawed aliquot of supernatant was used for each experiment and then discarded. Reaction products were analyzed by electrophoresis on a native 8% polyacrylamide gel in 0.5 × TBE (89 mm Tris, 89 mm boric acid, 4 mm EDTA, pH 8.3) at 4 °C. To eliminate hydrolysis of RNA during electrophoresis, gel solutions and running buffer were prepared in diethylpyrocarbonate-treated H2O, and the electrophoresis apparatus was washed frequently with detergent. Gels were dried and analyzed by autoradiography, and 32P-RNA was quantitated by PhosphorImager analysis on a Molecular Dynamics Imager model 425. Kapp values for mutant proteins were determined from gel shift assays in a manner similar to that of Rebar and Pabo (21Rebar E.J. Pabo C.O. Science. 1994; 263: 671-673Crossref PubMed Scopus (387) Google Scholar). Kapp. was calculated at four points on the titration curve (Equation 1). The mean of the four values was calculated, and Kapp values from 2–4 experiments were averaged. WT RegA in cell supernatants was assayed in parallel with 2–4 mutant proteins in each set of experiments. S.D. values (calculated using the nonbiased or “n − 1” method using EXCEL) ranged from Kapp (Av)/3.3 toKapp(Av)/11. Association constants were determined from phosphor image data as follows,Kapp=[protein−RNA complex]/([proteinf]×[RNAf])(Eq. 1) where [protein-RNA complex] = [fraction 32P-RNA bound] × [total RNA]; [RNAf ] = [fraction32P-RNA free] × [total RNA]; [proteinf ] = [total protein] − [bound protein], assuming one protein per bound RNA (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar). Circular dichroism spectra were collected on an Jasco spectropolarimeter model J-710 from 250 to 190 nm at 0.5-nm intervals. Spectra were recorded in 25 mm Tris, 25 mm NaCl, 5 mm MgCl2, 1 mm EDTA at 25 °C, using a 0.2-mm path length quartz cuvette. The data were averaged from 10 repeat scans and were corrected for background noise by subtraction of signal from buffer alone. Protein concentrations were determined by duplicate measurements ofA280, which had a error range of ±2% The molar extinction coefficients used were as follows: WT, K14A and T18A: εM = 22,250; W81A εM = 16,650. From inspection of the structure of RegA protein, Kang et al. (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar) noted that in domain I of RegA, basic residues in the loops at the base of the central two β-strands, i.e. Lys7, Lys8, Lys41, and Lys42 (see Fig.1) are located in positions similar to basic residues of U1A protein (residues Lys22, Lys23, Lys50, and Arg52) that function in RNA binding (12Oubridge C. Ito N. Evans P.R. Teo C-H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (784) Google Scholar). To test if these residues form electrostatic interactions with RNA, as they do in U1A, individual lysine to leucine substitutions were introduced at each of these residues to remove the basic charge and leave a similarly sized side chain. Mutant proteins K7L, K41L, and K42L were purified (see “Materials and Methods”), and their equilibrium RNA binding affinities were measured by fluorescence quenching assays (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar), as shown in Fig. 2. Like many other nucleic acid-binding proteins, the intrinsic tryptophan fluorescence of RegA protein is quenched upon binding to nucleic acids (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar). This quenching is due to a change in the environment of one or more of the three tryptophan residues in RegA, presumably reflecting either a direct interaction of the tryptophan(s) with RNA or a conformational change in RegA upon RNA binding. Because the fraction of the maximal quenching obtained is directly proportional to the fraction of RegA protein bound to RNA (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar), fluorescence quenching assays offer an accurate method of determining equilibrium binding constants. The affinities of mutant proteins for a specific RNA (T4 gene 44 RE RNA (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar)) and a nonspecific RNA (poly(U)) were assessed to evaluate the effect of these mutations on the RNA binding properties of RegA protein (Table I). As shown in Fig. 2 Aand Table I, all three purified mutant proteins bound gene 44 RE RNA with affinities similar to that of wild type (WT) RegA protein. In addition, K41L and K42L RegA proteins bound poly(U) with WT affinity (Table I); however, K7L bound poly(U) with approximately 10-fold lower affinity. Previous studies (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar) have indicated that RegA protein binds polynucleotides with weak cooperativity (i.e. with a cooperativity parameter (ω) of ∼10–12). Thus, the lower affinity of K7L for poly(U) could result from a decrease in RNA binding affinity or from alterations in protein-protein interactions leading to reduced cooperativity in RNA binding.Table IAffinity of purified WT and lysine-substituted RegA proteins for RNAProteinKappaBinding affinities were determined by equilibrium fluorescence quenching assays (4); S.D. values are given in those instances where multiple determinations were carried out. ± S.D. for gene 44 RNAbMeasured in buffer C plus 150 mm NaCl.Kapp ± S.D. for poly(U)cMeasured in buffer C plus 10 mm NaCl. Kapp calculation assumed the binding site size (n) = 9 (4).WT1.9 ± 0.8 × 1074.6 ± 0.8 × 107K7L1.1 ± 0.1 × 1074.2 ± 0.5 × 106K41L1.6 ± 0.3 × 1071.4 ± 0.5 × 107K42L2.8 × 1071.9 × 107a Binding affinities were determined by equilibrium fluorescence quenching assays (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar); S.D. values are given in those instances where multiple determinations were carried out.b Measured in buffer C plus 150 mm NaCl.c Measured in buffer C plus 10 mm NaCl. Kapp calculation assumed the binding site size (n) = 9 (4Webster K.R. Spicer E.K. J. Biol. Chem. 1990; 265: 19007-19014Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab To assess the effects of the mutations on electrostatic interactions between RegA and RNA, salt-back titrations (18McSwiggen J.A. Bear D.G. Von Hippel P.H. J. Mol. Biol. 1988; 199: 609-622Crossref PubMed Scopus (85) Google Scholar) were performed, in which NaCl was added incrementally to RegA protein-poly(U) complexes. As shown in Fig. 2 C, RNA complexes formed with mutated proteins K41L and K42L exhibited salt sensitivities similar to that of WT RegA protein, while K7L-poly(U) complexes were considerably more sensitive to ionic strength. These data suggest the observed reduction in Kapp for nonspecific poly(U) (TableII) may be due to decreased protein affinity for RNA, rather than decreased cooperativity, since RegA protein-protein interactions have been found to be unaffected by salt concentrations (150 mm NaCl) that disrupted the K7L RNA complexes (7Phillips C.A. Gordon J. Spicer E.K. Nucleic Acids Res. 1996; 24: 4319-4326Crossref PubMed Scopus (14) Google Scholar).Table IIAffinity of domain I mutant RegA proteins for gene 44 RE RNAMutant proteinKappaKapp was determined by quantitative RNA gel shift analysis (see “Materials and Methods”). Binding was performed in 10 mm Tris (pH 7.5), 50 mm NaCl, and 1 mm EDTA. Values are the mean of 2–4 experiments except for WT (nine experiments). ± S.D.Location of residuebAll mutated residues are located on the surface, except for Tyr46, where the OH group is exposed, but most of the aromatic ring is in the interior.107m−1Wild type4.7 ± 0.9I4A2.7 ± 0.3β1T5A3.1 ± 0.2β1L6R5.7 ± 1.5β1K7L4.8 ± 1.2Loop 1K8L2.1 ± 0.1Loop 1E10A14.7 ± 2.5α-Helix AD11A13.1 ± 0.5α-Helix AF12A2.4 ± 0.2α-Helix AK14A0.7 ± 0.1α-Helix AK16L1.7 ± 0.1α-Helix AE17A2.8 ± 0.1α-Helix AT18A0.15 ± 0.01α-Helix AR21A—c—, no binding observed.α-Helix AK41L4.9 ± 0.5β4K42L2.6 ± 0.1(Hairpin) loop 4L44K3.2 ± 0.2β5Y45A3.3 ± 0.2β5Y46A1.3 ± 0.1β5W76A4.8 ± 0.1α-Helix CW81A—Loop 6L83A7.4 ± 0.8Loop 6F93Y5.4 ± 0.2β6Residue locations are from accession number 1REG (C. Kang and A. Rich) of the Protein Databank at Rutgers University; however, the numbering of β-strands and α-helices corresponds to assignments given in Kanget al. (6Kang C.-H. Chan R. Berger I. Lockshin C. Green L. Gold L. Rich A. Science. 1995; 268: 1170-1173Crossref PubMed Scopus (21) Google Scholar). Italic type indicates ≥6-fold reduction inKapp; boldface type indicates loss of RNA binding.a Kapp was determined by quantitative RNA gel shift analysis (see “Materials and Methods”). Binding was performed in 10 mm Tris (pH 7.5), 50 mm NaCl, and 1 mm EDTA. Values are the mean o
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