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• W2076228729 abstract "The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of >12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 1→94 and 1→109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding. The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of >12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 1→94 and 1→109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding. The bacteriophage T4 regA protein regulates the expression of at least 12 T4 genes, while also regulating its own synthesis at the level of translation (for review, see Miller et al.(1994)). It has been shown that regA protein acts by binding to specific mRNAs and competing with the formation of initiation complexes by ribosomes (Winter et al., 1987; Unnithan et al., 1990). We have previously shown that repression of T4 gene 44 by regA protein involves the recognition of a specific RNA element, in which apparently both the sequence and structure are of importance (Webster and Spicer, 1990; Szewczak et al., 1991). More recent studies have focused on structure-function relationships in regA protein in an effort to identify domains and specific amino acid residues involved in RNA recognition and binding (Webster et al., 1992; Jozwik and Miller, 1992). Photochemical cross-linking has identified amino acid residues at the interface of a regA protein: nucleic acid complex (Webster et al., 1992). Two sites of cross-linking to p(dT)16 were identified: the major site of cross-linking was at Phe-106 and the minor site was the peptide spanning 31→41, with the site of cross-linking tentatively assigned to Cys-36 (see Fig. 1). A COOH-terminal peptide (containing residues 95→122) produced by CNBr cleavage of regA protein was found to retain the ability to bind to nonspecific RNA and to be photo-cross-linked to p(dT)16 (Webster et al., 1992). In addition, Jozwik and Miller(1992) have examined mutations in T4 and the related phage RB69 regA proteins and have found that two regions are particularly sensitive to mutations: the regions from Val-15 to Ala-25 and between Arg-70 and Ser-73. Finally, amino acid similarities to other RNA-binding proteins have been identified for two regions of regA protein; one in the NH2-terminal region between residues Val-15 to His-37 containing a potential helix-turn-helix structural motif (Jozwik and Miller, 1992) and the other in the COOH-terminal region between residues Leu-97 and Lys-113 (Webster et al., 1992) (see Fig. 1). In this study, the role of residues in the COOH-terminal region of regA protein have been examined further. In order to determine if residue Phe-106, the major site of photo-cross-linking, plays a significant role in RNA-protein interactions, site-specific mutagenesis was used to introduce three substitutions at codon 106. To assess the role of residues neighboring Phe-106, two truncation mutants were constructed containing residues 1→109 and 1→94 (Fig. 1). In addition, we have examined the sensitivity of regA protein to partial proteolysis by chymotrypsin and trypsin. Poly(U), poly(U)-hexane agarose, and p(dT)16 were purchased from Pharmacia Biotech Inc. All other oligoribonucleotides were synthesized by the Keck Foundation Biotechnology Resource Laboratory (Yale University). Deprotection and high performance liquid chromatography purification of synthetic RNAs was performed as described by Webster et al.(1991). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs Inc. Sequencing grade chymotrypsin and trypsin were purchased from Boehringer Mannheim. [γ-32P]ATP was obtained from Amersham Corp. N-Acetyl-L-tryptophanamide was purchased from Sigma Oligonucleotide-directed mutagenesis was carried out by annealing mutagenic oligonucleotides to single-stranded DNA of a phage M13 derivative carrying the wild type regA gene (Webster et al., 1989). The following primers were used: for Phe-106 mutations, 5′-CCATTCATGTTTTTCTTTAN(C/A)AGAAATAACTCGG-3′; fragment 1→94, 5′-GTCAAAGAACTTTTATGTAAGCTTTAACTAATAACTTCCG-3′; fragment 1→109, 5′-TTCTTTTAAACAAAAATAAGCTTGGAAGCTCGTTCCTAAA-3′. Primer annealing, chain extension, and transfection of Escherichia coli was carried out as described by Sambrook et al.(1989). Mutations were confirmed by DNA sequence analysis, using an ABI 373A automated DNA sequencer. WT 1The abbreviations used are: WTwild typePAGEpolyacrylamide gel electrophoresisLDMSlaser desorption mass spectrometryRNPribonucleoprotein. and mutant regA proteins were purified from AR120 cells containing plasmid pAS1regA, following induction of transcription from the phage λPL promoter by nalidixic acid treatment (Webster and Spicer 1990). Purification of WT and Phe-106 mutant regA proteins was as described previously by Adari and Spicer(1986). Both deletion mutants 1→94 and 1→109 were found to be insoluble under normal induction conditions. For fragment 1→109, lowering the induction temperature from 37 to 25°C enhanced the solubility to approximately 80%. Proteolysis during purification was retarded by maintaining the level of active phenylmethylsulfonyl fluoride at 1 mM. The regA protein fragment 1→109 demonstrated a low affinity for poly(U)-agarose. Accordingly, the flow-through fractions from DE52 chromatography were applied to a mono S column. The 1→109 fragment was eluted at approximately 0.4 M NaCl and was dialyzed against buffer Uo (20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol) plus 20 mM NaCl, to remove excess salt prior to final chromatography on poly(U)-agarose. The deletion mutant 1→94 was found to be efficiently resolubilized from the membrane fraction by stirring the pelleted material in lysis buffer (20 mM Tris-HCl (pH 7.5) 100 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), at 4°C overnight. The resolubilized 1→94 protein was then purified by DE52 and poly(U)-agarose column chromatography. wild type polyacrylamide gel electrophoresis laser desorption mass spectrometry ribonucleoprotein. WT and mutant regA proteins [10 μM] were irradiated in the presence of 15 μM [32P](dT)16 in 25 μl of buffer A (10 mM Tris, 75 mM NaCl, 2.5 mM MgCl2, 0.5 mM dithiothreitol, 5% glycerol, pH 7.5). Samples were irradiated with a germicidal UV lamp (253 nm) at a height of 5 cm (generating 5 × 103 ergs/mm2/min) at 4°C in a Petri dish. Cross-linked products were detected by SDS-PAGE and the efficiency of cross-linking was estimated by densitometry of autoradiograms using Adobe Photoshop and NIH Image programs. The fluorescence of WT and mutant regA proteins were detected on an SLM 8000C spectrofluorometer, interfaced with an IBM PC-XT computer. Titrations were performed in a quartz cuvette with continuous stirring, at 25°C. Binding titrations were measured in 2.0 ml of buffer C [10 mM Hepes (pH 7.2), 5 mM MgCl2, 1 mM EDTA, 1 mM β-mercaptoethanol] plus a specified concentration of NaCl. All protein concentrations were based on duplicate amino acid analysis. Protein fluorescence was monitored at a fixed excitation wavelength of 282 nm and an emission wavelength of 347 nm. Excitation and emission bandpasses were set to 8.0 nm. All fluorescence binding data were acquired through “reverse” titrations (the addition of nucleic acid lattice to protein ligand). All fluorescence values were corrected for background fluorescence, dilution effects, protein photo-bleaching and inner filter effects due to the intrinsic absorbance of nucleic acids. Inner filter corrections were derived from third-order regression of data obtained from titrating N-acetyl-L-tryptophanamide and the appropriate polynucleotide. The effect of salt concentration on RNA binding affinity was determined by a salt back titration (McSwiggen et al., 1988), using successive additions of 5 M NaCl. The apparent association constant (Kapp) was calculated using the equivalence point of the titration and a binding site size of n = 9 residues (assuming non-overlapping sites) for regA protein binding to polynucleotides, as determined previously (Webster and Spicer, 1990). Direct determination of the binding site size for regA protein fragments 1→94 and 1→109 binding to poly(U) was not possible because of the low affinities of the proteins for nucleic acids. A simplifying assumption was made that the site size for both protein fragments is likely to be smaller than that of WT regA protein since the fragments correspond to approximately two-thirds the size of regA protein. Accordingly a proportional site size of 6 nucleotides was assumed. If the occluded site sizes of the deletion mutants were 9 nucleotides, then the Kapp would be 2-fold higher than estimated. For oligonucleotide gene 44-4, a single binding site was assumed for both regA protein and the protein fragments. The maximal percent quenching (%Qmax) was determined by two methods (a) the observation of a constant plateau in protein fluorescence quenching after successive additions of lattice to regA samples or (b) for oligonucleotide gene 44-4, in cases were a stable plateau was not reached, %Qmax was derived from double-reciprocal analysis in which 1/ΔF was plotted versus 1/(free oligo) (Kelly et al., 1976). The y intercept of the plot yields 1/Δ Fmax. Circular dichroism spectra were collected on an Aviv circular dichroism spectropolarimeter model 62DS from 300 to 200 nm at 0.5-nm intervals, using a 1-mm quartz cuvette. All protein spectra were recorded in buffer C plus 75 mM NaCl, at 25°C. The data averaged from four repeat scans was fit to a smoothing polynomial function. The protein spectra were corrected for background noise present in buffer alone. Following analysis, protein concentrations (5-10 μM) of each individual sample were determined by duplicate amino acid analyses which have an average error of about ±10%. The distribution of secondary structures was estimated from spectra extending from 204-240 nm by the Prosec algorithm of Aviv (Chang et al., 1978). We note that, while this approach generally provides a good estimate of the fraction of α-helix (Johnson, 1988), in some proteins it may give errors in estimates of secondary structure elements that exceed 20%, and it may be unable to adequately distinguish between β-sheet versus β-turn structures (Chang et al., 1978). Hence, we have not attempted to differentiate between the latter two structures. WT and mutant regA proteins (14 μM protein in 20 mM Tris (pH 8)) were digested by trypsin or chymotrypsin, at a 25:1 (w/w) ratio of regA protein to protease, for various times, at 37°C. To purify the 11-kDa chymotryptic fragment, 1 mg of WT regA protein was digested for 30 min, after which proteolysis was stopped by addition of diisopropyl fluorophosphate (final concentration, 1 mM). Digestion products were then purified by chromatography on poly(U)-agarose and DEAE-cellulose (Adari and Spicer, 1986). The effects of RNA binding on partial proteolysis of regA protein was assessed by performing parallel digestions in the presence and absence of an equimolar amount of the 16-mer target RNA gene 44-4. For NH2-terminal sequence analysis of the 11-kDa fragment, the products of a chymotryptic digest were separated on an SDS-17.5% polyacrylamide gel and then transferred onto an Immobilon P membrane (Millpore). Amino-terminal sequencing was then performed directly on the membrane section containing the 11-kDa chymotryptic fragment. Laser desorption mass spectrometry (LDMS) analyses were performed on a VG TOFSPEC (Fisons) mass spectrometer. For determination of tryptic and chymotryptic peptide masses, the mass spectrometer was scanned from m/z = 500 to m/z = 17000. The samples were run in an α-cyano matrix. The instrument was calibrated using β-lactoglobulin (18,281 Da and its doubly charged ion, 9,141 Da), insulin (5,734 Da), and gramicidin (1,142 Da). All LDMS analyses were performed by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Samples submitted to LDMS analysis were prepared by 10% trichloroacetic acid precipitation, as described by Stone et al.(1989). The precipitate was then resuspended in deionized H2O. Oligonucleotide-directed mutagenesis was carried out using degenerate oligonucleotides (see “Material and Methods”) and a previously constructed phage M13 derivative carrying the WT regA gene (Webster et al., 1989). Three clones with nucleotide changes coding for substitutions of Val, Tyr, and Cys at Phe-106 were identified by DNA sequence analysis. Restriction fragments carrying the regA gene from the three mutant clones were subcloned into the inducible expression vector pAS1 (Rosenberg et al., 1983; Adari et al., 1985). The mutant proteins were produced at high levels by nalidixic acid induction of expression from the pAS1-regA vector (Mott et al., 1985; Adari et al., 1985). All three proteins were purified using a previously described method of chromatography on DEAE-cellulose and poly(U)-agarose (Adari and Spicer, 1986). The effects of the amino acid substitutions on the overall conformation and functional activity of regA protein were then assayed by circular dichroism spectroscopy and by photo-cross-linking and fluorescence quenching assays. Comparison of the CD spectra of the mutant proteins with that of wild type regA protein, shown in Fig. 2A, indicate that substitutions of Cys and Tyr for Phe-106 do not produce large alterations in the protein's secondary structure. Based on variations observed in the 200-215-nm region of repeated CD scans of WT regA protein (Fig. 2B), the variations in CD spectra observed for the F106C and F106Y proteins are relatively small. Although the difference between the F106V and the WT scan appears larger than that of the other two mutants, we note that the shape of the F106V and WT scans are extremely similar and that the difference in absolute molar ellipticity of these two proteins is less than the ±10% average error in determining their concentrations (see “Materials and Methods”). Prosec analysis of these spectra generally confirms these qualitative conclusions. As shown in Table 1, the F106Y and F106V mutants have predicted α-helical contents that are within two standard deviations of the range seen for the WT protein. In the case of F106C, Prosec analysis predicts this substitution decreases the α-helical content by less than 8%. That substitution of Phe-106 by these three mutations does not result in any large change in the protein's secondary structure is further confirmed by limited proteolysis studies. That is, comparative partial proteolysis using either chymotrypsin or trypsin (see below for proteolysis of WT regA protein) did not reveal any observable differences in sensitivity to proteolysis, consistent with the overall conformations of the three substitution mutants being similar to that of WT.Table I Open table in a new tab As a first step toward determining if the mutant proteins bind to nucleic acids in a manner similar to that of wild type regA protein, the ability of the proteins to be photo-cross-linked to [32P]p(dT)16 was examined. As shown in Fig. 3, all three mutant proteins retain the ability to be photo-cross-linked to p(dT)16, although with variable efficiency. As judged by gel electrophoresis and densitometry of autoradiograms, the efficiency of cross-linking of the proteins with the three substitutions was Cys > Phe > Val > Tyr with corresponding efficiencies of 1.2:1.0:0.7:0.3. The variation in efficiencies presumably is due to differences in reactivity of the four amino acids and may also be a reflection of changes in the topology of protein:DNA contacts. To further examine the functional activity of the mutant proteins, the ability of the proteins to inhibit translation of three T4 genes was examined. For these assays, plasmid pTL45W, which contains T4 genes rpbA, 45, and the 5′ half of gene 44 (Δg44) under the transcriptional control of the λPL promoter, was used as a template in in vitro coupled transcription-translation assays (see Webster et al., 1989). Based on these studies (data not shown), it appears all three Phe-106 mutant regA proteins retain the ability to inhibit translation of the three regA-controlled genes on this plasmid. To quantitatively assess the effect of the Phe-106 amino acid substitutions on the functional activity of regA protein, nonspecific and specific RNA binding affinities were measured. Fluorescence quenching assays were used to measure the apparent equilibrium binding constants (Kapp) of the three mutants for poly(U) and for the gene 44-4 16-mer RNA (5′-AAUGAGGAAAUUAUGA-3′), corresponding to the binding site for regA protein on gene 44 mRNA (Webster and Spicer, 1990). The reverse fluorescence titration curves, shown in Fig. 4A, demonstrate that the affinity of the Phe-106 regA substitution mutants for the nonspecific RNA target poly(U) is very similar to that of wild type regA protein. The corresponding salt-back titrations for the Phe-106 substitution mutants, shown in Fig. 4B, indicate that binding of all three mutant proteins is slightly more sensitive to ionic strength than WT regA protein binding. Reverse titrations for specific binding to the target gene 44-4 RNA, shown in Fig. 5, demonstrate binding affinities that are, again, very similar to that of the wild type regA protein. The affinity constants derived from these binding curves, given in Table 2, indicate that the substitutions at Phe-106 do not significantly affect the ability of regA protein to bind to either nonspecific or specific RNA.Figure 5:Reverse titrations of Phe-106 substituted mutants and wild type regA protein with specific target RNA, gene 44-4 (16 mer). Titrations were performed at 0.2 μM protein in buffer C plus 150 mM NaCl (25°C). Symbols used are the same as in Fig. 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table II Open table in a new tab Using in vitro mutagenesis (as described above), two mutant regA genes were constructed with stop codons introduced at positions Lys-95 and His-110 (see Fig. 1). Expression of the resultant deletion mutants (1→94 and 1→109) from the expression vector was very efficient; however, both proteins were prone to the formation of insoluble aggregates (inclusion bodies). The 1→94 regA protein deletion mutant was successfully resolubilized by stirring for several hours at 4°C. The solubility of the 1→109 fragment was found to be increased to approximately 80% by carrying out induction of regA protein synthesis at 25°C instead of 37°C. To evaluate whether the fragments were folded in a near native conformation, CD spectra were measured for the deletion mutants (Fig. 6A). The CD spectra of a synthetic peptide corresponding to COOH-terminal residues 95→122 was also measured (Fig. 6B). Summation of the CD spectra of the 1→94 fragment and the synthetic peptide 95→122 resulted in a composite CD spectra (Fig. 6B) which was found to overlap closely with that of the unmodified wild type protein. Prosec analysis of these spectra suggest that while the 95-122 synthetic peptide contains no detectable α-helix the 1→94 fragment contains 57%α-helix. The latter corresponds to about 54 residues of α-helix, as opposed to the ~66 residues predicted in the native protein (Table 1). Thus, while cleavage at Met-94 may lead to the loss of ~12 residues of α-helical structure, it does not appear to substantially alter the overall regA protein secondary structure. To evaluate the RNA binding affinities of regA proteins 1→94 and 1→109, reverse titration fluorescence quenching assays were performed. As illustrated in Fig. 7, the observed fluorescence quenching of 1→109 and 1→94 upon poly(U) binding were both dramatically reduced relative to that of WT regA protein (Fig. 4A). The estimated %Qmax for poly(U) titrations of 1→94 and 1→109 were 16 and 35%, respectively. This reduction in %Qmax, from the 75% value observed with WT regA protein, suggests that the individual contributions of the three tryptophans (Trp-78, Trp-81, and Trp-112) to regA protein fluorescence quenching may not be equal and that Trp-112 may be a major contributor to the fluorescence quenching observed upon poly(U) binding. The reduced level of fluorescence quenching and weaker binding (see below) observed for the regA protein fragments made estimation of %Qmax and subsequent calculation of Kapp somewhat less accurate than for regA protein. Nevertheless, approximations of Kapp could be made from double reciprocal plots (for gene 44-4 oligonucleotide) or from curve fitting (poly(U)). The Kapp derived from these analysis, given in Table 3, indicated that the binding affinities of 1→94 and 1→109 for poly(U) and gene 44-4 RNA are approximately 100-1000-fold lower than that of wild type regA protein.Table III Open table in a new tab To further probe structural and functional domains of regA protein, the susceptibility of regA protein to proteolytic cleavage was examined. Digestion of regA protein with either trypsin or chymotrypsin produced a stable core fragment of ~11 kDa as observed by SDS-PAGE (Fig. 8). When chymotryptic digestion was carried out in the presence of gene 44-4 RNA, the rate of hydrolysis by chymotrypsin was significantly decreased. Thus, after 15 min of digestion in the absence of RNA, approximately 90% of regA protein was converted to the 11-kDa species, while in the presence of RNA, roughly 20% of the protein was cleaved. When the products of the chymotrypsin digestion were applied to a poly(U)-agarose column essentially all of the 11-kDa fragment was retained. However, the fragment was eluted from the column at 0.3 M NaCl, while WT regA is completely eluted by 0.8 M NaCl. Thus, it appears that the chymotryptic fragment has a significantly reduced affinity for RNA. This was confirmed by fluorescence quenching titrations with gene 44-4 RNA (Table 3). To determine the site(s) of chymotryptic cleavage that produced the 11-kDa fragment, fragments eluted from a poly(U) agarose column were separated by SDS-PAGE and then electro-transferred to an Immobilon P membrane. NH2-terminal micro-sequencing was performed on the membrane section containing the 11-kDa polypeptide. The sequence of the first 4 residues revealed that the fragment has the same NH2-terminal sequence as the intact protein. Determination of the masses of the chymotryptic and tryptic core fragments by LDMS revealed that the chymotryptic fragment (11,095 Da) (Fig. 9) is approximately 200 daltons larger than the tryptic fragment (10,898 Da). These mass determinations and NH2-terminal sequencing data together indicate that the site of cleavage by chymotrypsin producing the 11-kDa fragment, is at Phe-93, while that of trypsin is at Arg-91. In addition, LDMS analysis of chymotryptic digestion products indicated 4-5 smaller peptide masses (see Fig. 9), indicating that multiple proteolytic cleavages occur in the COOH-terminal region. The possible sites of chymotrypsin cleavage (after aromatic residues) in this region are at Phe-93, Phe-101, Phe-106, Trp-112, and Tyr-118. As shown in Table 4, four of the observed chymotryptic fragments have masses corresponding to peptides predicted from cleavages at three of these sites (i.e. after Phe-93, Phe-106, and Tyr-118). While these cleavages may occur after a COOH-terminal fragment has been released from regA protein, the observation of two intermediate proteolytic products by SDS-PAGE (Fig. 8) suggests that these cleavages can occur in the intact protein. Taken together with the CD studies, these results suggest the NH2-terminal three quarters of regA protein are folded into a structured, protease resistant core, while the COOH-terminal one quarter is significantly more flexible and exposed to solvent.Table IV Open table in a new tab The functional activities of three regA protein mutants with substitutions at Phe-106 were assayed by UV-induced photo-cross-linking and fluorescence titrations of RNA binding. regA F106C and F106V proteins were found to retain a near native ability to be photo-cross-linked to p(dT)16, while F106Y demonstrated a reduced cross-linking efficiency. Coupled with the negligible effect of the F106Y mutation on RNA binding affinity, this result suggests that the preponderance of proteins that have been found to be cross-linked at Phe (Williams and Konigsberg, 1991), as opposed to Tyr, results from the unique ability of Phe to participate in these photochemical reactions, as opposed to any unique ability of Phe to participate in interactions with single-stranded nucleic acids. The nonspecific and specific RNA binding affinities of all three mutants were found to be very similar to that of wild type regA protein. However, RNA binding of all three substitutions was found to be slightly more salt sensitive than wild type regA protein suggesting slight changes in the types of RNA: protein interactions. Previous fluorescence binding studies (Webster and Spicer, 1990) demonstrated that RNA recognition by WT regA is weakly salt-sensitive and that as few as two ionic interactions may be involved in RNA binding by WT regA protein. Taken together, these results indicate that while Phe-106 is at the interface of a regA protein-nucleic acid complex, it does not contribute significantly to the overall free energy of RNA binding. Partial proteolysis studies suggest that the NH2-terminal three quarters of regA protein are folded into an ordered, protease-resistant core, while the COOH-terminal one quarter is more exposed to solvent and/or less structured. The crystal structure of regA protein solved by C.-H. Kang, A. Rich and co-workers 2C.-H. Kang, R. Chan, I. Berger, C. Lockshin, L. Green, L. Gold, and A. Rich(1995), manuscript submitted for publication. reveals that residues 90-96, as well as a number of additional COOH-terminal residues, are on the surface of the protein. Interestingly, RNA binding to regA protein results in a reduction in susceptibility to proteolytic cleavage. This is presumably due either to a direct shielding of the chymotryptic cleavage sites on regA protein by the bound RNA or to a conformational change in the COOH-terminal region induced by RNA binding. Such a reordering of an unstructured region has been proposed for a region of the RNP domain of U1 sn-RNP A protein (Nagai et al., 1990) and has been observed in a number of other nucleic acid binding proteins (Steitz, 1990). Previous studies (Webster et al. 1992) demonstrated that a COOH-terminal fragment of regA protein (CNBr 6) containing residues 95-122 retained the ability to be photo-cross-linked to p(dT)16 and to bind poly(U) (Kapp = 3 × 104M−1 in 10 mM NaCl). To further evaluate the relative contribution of COOH-terminal residues to RNA binding, a complimentary fragment containing residues 1-94 was produced and tested for RNA binding. This fragment has an affinity of approximately 3 × 104M−1 for poly(U) (in 10 mM NaCl) (Table 3). Thus, both fragments have weak affinities for RNA. Summation of the free energies of RNA binding for the two fragments is somewhat higher than the free energy for intact regA protein binding to poly(U). That is, the free energy of regA protein binding is ~10.0 kcal, compared to ~12.2 kcal for the sum of the free energies of binding of the two fragments. This discrepancy may arise from inaccuracies in estimation of the binding site size and/or %Qmax for both fragments. Also, the CNBr6 peptide may bind somewhat tighter when it is free in solution than as part of intact regA protein (see Nadler et al., 1992). These binding studies indicate that the NH2-terminal 1→94 fragment of regA protein retains the ability to bind to RNA, although with significantly weaker affinity than intact regA protein. The 1→109 fragment, containing 15 additional C-terminal residues (including Phe-106) had a somewhat higher affinity for poly(U) than 1→94. However, the 1→109 fragment binds RNA with a 100-fold lower affinity than WT regA protein, indicating that a large part of the fragments' observed defects in RNA binding is due to the absence of the COOH-terminal 13 residues. Interestingly, this region was identified by Jozwik and Miller(1992) as being one of three mutationally sensitive regions of regA protein. Based on the relative affinities of 1→109 and WT regA protein for poly(U) (2 × 105versus 2 × 107M−1), the C-terminal 10% of regA protein contributes approximately 28% of the overall free energy of RNA binding. Thus, although other regions of regA protein also must play important roles in RNA binding, the proteolysis and truncation studies reported here demonstrate that COOH-terminal residues make significant contributions to RNA:regA protein interactions. The crystal structure of regA protein2 has revealed similarities between NH2-terminal residues of regA protein and the RNP domain found in a number of RNA-binding proteins (Bandziulis et al., 1989), including the U1 sn-RNP A protein (Nagai et al., 1990). We anticipate that future mutagenesis and x-ray structural studies of regA protein will reveal how both the NH2- and COOH-terminal regions of regA protein are associated with RNA in protein-RNA complexes. We are grateful to William Konigsberg for his continuing interest in this work. We thank John Lee for assistance with purification of truncation mutants, Yvonne Bernie for purification of F106V regA protein, and Dr. Lynn Reagen for use of the circular dichroism spectropolarimeter. We gratefully acknowledge the skillful DNA sequencing, protein chemistry, and LDMS analyses performed by the W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University). We also thank Ann Ewing and Burnett G. Bryant for careful preparation of this manuscript." @default.
• W2076228729 created "2016-06-24" @default.
• W2076228729 creator A5023101057 @default.
• W2076228729 creator A5052344044 @default.
• W2076228729 creator A5064900587 @default.
• W2076228729 creator A5066944549 @default.
• W2076228729 date "1995-03-01" @default.
• W2076228729 modified "2023-09-26" @default.
• W2076228729 title "Mutagenesis of the COOH-terminal Region of Bacteriophage T4 regA Protein" @default.
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• W2076228729 doi "https://doi.org/10.1074/jbc.270.10.5107" @default.
• W2076228729 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7890619" @default.
• W2076228729 hasPublicationYear "1995" @default.
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