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Journal of Bacteriology, February 2003, p. 801-808, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.801-808.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role for both DNA and RNA in GTP Hydrolysis by the Neisseria gonorrhoeae Signal Recognition Particle Receptor

Cody Frasz and Cindy Grove Arvidson*

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1101

Received 25 June 2002/ Accepted 13 November 2002


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ABSTRACT
 
The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that PpilE DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.


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INTRODUCTION
 
Proper targeting of proteins synthesized in bacteria is an essential and complex process. In gram-negative bacteria, about 20% of synthesized proteins are exported out of the cytoplasm to one of four different compartments: the cytoplasmic membrane (CM), the periplasm, the outer membrane, or the extracellular milieu. Most exported proteins are initially targeted to the CM via one of two pathways, the general secretory pathway (reviewed in references 44, 49, and 60) or the signal recognition particle (SRP) pathway (5, 55). The general secretory pathway is a posttranslational targeting system used by a variety of exported proteins, while the SRP functions cotranslationally to target a subset of proteins whose final destination is the CM (11, 31, 37, 51, 55, 56). The use of a cotranslational targeting system by this subset of proteins is likely advantageous in that it avoids their aggregation in the cytoplasm, which might occur if they were targeted posttranslationally.

The components of the prokaryotic SRP were initially identified on the basis of their homology to proteins of the well-characterized eukaryotic SRP (6, 46), a ribonucleoprotein (RNP) complex of six different polypeptides and a 7S RNA that is required for the targeting and insertion of exported proteins into the endoplasmic reticulum membrane (45, 59). The prokaryotic SRP pathway is much simpler than the eukaryotic counterpart, consisting of just two proteins, Ffh and FtsY, and a 4.5S RNA, all of which are essential for viability in Escherichia coli (8, 16, 40). Orthologs of the SRP components appear to be present in all prokaryotes for which sequence information is available, and the SRP genes have been suggested to belong to the minimal set of genes required for independent life (18).

Ffh is the prokaryotic homolog of SRP54, the eukaryotic SRP signal sequence-binding protein, hence the name Ffh (fifty-four homolog). SRP54 has one of the key functions of the SRP, recognition of the signal sequence of the nascent polypeptide (63). Ffh is composed of three domains. The methionine-rich M domain interacts with the signal peptide of nascent proteins, as well as the 4.5S RNA (4, 7, 62). The G domain has the GTPase activity that is required for its interaction with the docking protein and the subsequent release of the nascent peptide at the translocon (48). Finally, the highly conserved N domain is thought to sense and/or control the GTP occupancy of the G domain (15). FtsY, the membrane-associated SRP receptor or docking protein, also has three domains. The G domain of FtsY is homologous to the G domain of Ffh and has a similar function (32). Similarly, the N domain of FtsY is homologous to the N domain of Ffh. The negatively charged amino-terminal A domain of FtsY is important for membrane localization (34, 42, 61) and may also be involved in modulating the GTPase activity in response to the interaction with the CM (13). Ffh and FtsY are both GTPases (3, 24, 43), and their GTPase activities are required for their function in protein targeting (24, 42, 48).

SRP-dependent proteins have a hydrophobic amino-terminal signal sequence that is recognized by the Ffh-4.5S RNA RNP complex (29, 32). Several reports suggest that the hydrophobicity of the signal sequence is an important factor in determining whether a protein will use the SRP for targeting (12, 26, 57). It has also been suggested that trigger factor, a ribosome-associated chaperone, plays a role in the SRP-Sec decision by competing for binding to the signal sequence of the nascent polypeptide chain as it emerges from the ribosome (5); however, the role of trigger factor has not been demonstrated in other studies. Once the RNP binds the signal sequence, the complex (RNP-nascent peptide-ribosome) is targeted to the membrane-associated SRP docking protein or receptor, FtsY. Following binding to FtsY, the nascent protein is transferred from the SRP to the SecYEG translocon concomitant with Ffh- and FtsY-catalyzed GTP hydrolysis and the protein is inserted into the membrane as translation continues (58). While it is clear that the signal for targeting to the SRP pathway is in the amino terminus (signal sequence) of the nascent peptide (12, 26, 57), another study has shown that a large periplasmic domain of the bitopic CM protein, AcrB, was necessary for its targeting via the SRP (38). Thus, it appears that there may be multiple factors involved in the targeting of a protein to the SRP apparatus.

We have previously shown that Neisseria gonorrhoeae PilA is a homolog of FtsY and is therefore the docking protein or receptor for the gonococcal SRP (1). We have also shown that PilA has sequence-specific DNA-binding activity in vitro (2). Up to this point, we have not been able to reconcile the DNA-binding capability of PilA with its role in protein targeting. In this work, we characterize the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and examine interactions among the three SRP components in vitro. We also investigate the DNA-binding activity of PilA and suggest a role for this activity in protein targeting via the SRP.


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MATERIALS AND METHODS
 
Growth and construction of bacterial strains. The E. coli strains used were JM109, DH5{alpha}, and BL21{lambda}(DE3) (52). E. coli cells were routinely grown in Luria broth supplemented as necessary with ampicillin or carbenicillin at 100 mg/liter or with kanamycin at 60 mg/liter. The N. gonorrhoeae strains used were derivatives of MS11A (P+tr) (50) and were maintained in a humidified 5% CO2 atmosphere on GC agar (Difco Laboratories, Sparks, Md.) with supplements (23).

DNA manipulations. E. coli recombinant DNA manipulations were done as previously described (47). The cloning vectors used were pET24a (Novagen, Madison, Wis.) and pBluescript II SK- (Stratagene, La Jolla, Calif.). Restriction enzymes (New England Biolabs, Beverly, Mass.), T4 DNA ligase, and polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, Ind.) were used in accordance with the manufacturers' recommendations. PCR was done with a 96-well GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, Calif.) and Taq DNA polymerase (Roche). Oligonucleotide primers were purchased from Sigma-Genosys (The Woodlands, Tex.). DNA sequence determination was done by the Core Facility of the Department of Molecular Microbiology and Immunology at Oregon Health and Science University with an ABI 377 automated fluorescence DNA sequencer (Applied Biosystems). DNA sequences were analyzed with the Oxford Molecular Group DNA analysis programs MacVector and Omiga (Accelrys, San Diego, Calif.).

Cloning of ffh and ffs. Oligonucleotide primers homologous to the 5' (CGA66) and 3' (CGA67) ends of the ffh open reading frame (as identified in the genome sequence of N. gonorrhoeae strain FA1090 [B. A. Roe, S. P. Lin, L. Song, X. Yuan, S. Clifton, and D. Dyer, Gonococcal Genome Sequencing Project, 2002; http://www.genome.ou.edu/gono/htm]) were used to PCR amplify ffh from total genomic DNA of MS11A. Each of the PCR primers included a unique restriction site designed specifically for cloning into expression vector pET24a (Novagen); an NdeI site at the 5' end included the start codon (ATG) of ffh, and a HindIII site in the 3' primer included the stop codon (TAA). The PCR product was digested with NdeI and HindIII and inserted into similarly digested pET24a to yield pETFfh. DNA sequences were determined from three independent clones from three separate PCR amplifications to rule out the possibility of mutations introduced by PCR. One clone, pETFfh-2, differed from the others at nucleotide position 112 (C->T). This was a silent mutation, resulting in no amino acid change. All three clones differed from the FA1090 sequence at nucleotide position 1347 (A->G), resulting in an amino acid change of isoleucine to methionine (Fig. 1). To clone ffs, oligonucleotide primers homologous to the 5' and 3' ends of the N. gonorrhoeae strain FA1090 ffs gene (CGA76 and CGA77; Fig. 2) were designed on the basis of the sequence in the SRP database (SRPDB) (17; C. Zwieb, Signal Recognition Particle Database, 2001; http://psyche.uthct.edu/dbs/SRPDB /SRPDB.html). The ffs gene was isolated by reverse transcription (RT)-PCR with low-molecular-weight RNA from strain MS11A as a target. A band of the predicted size, 131 bp, was produced following PCR when cDNA or genomic DNA from either strain (MS11A or FA1090) was used. Products from the RT-PCRs were excised from polyacrylamide gels, and the DNA was purified, digested, and ligated into pBluescript II SK- (Stratagene) such that ffs was under the control of the T7 promoter. Resulting clones were sequenced and found to be identical to each other and to the sequence reported in the SRPDB (also shown in Fig. 2). One clone, pBlu-ffsM13, was selected and used as a template for in vitro transcription (IVT) to produce 4.5S RNA.



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  		FIG. 1. Predicted amino acid sequence of MS11A Ffh. The single difference between the MS11A sequence and that of N. gonorrhoeae strain FA1090 (GenBank accession no. AE004969) is a methionine at position 449 in MS11A (underlined and in bold) that is an isoleucine in FA1090. The amino-terminal 15 amino acids determined by protein sequencing are double underlined. The amino-terminal 15 amino acids (beginning at residue 51) of the proteolytic fragment (indicated by the question mark in Fig. 3) are single underlined. Residues comprising the three motifs of the highly conserved GTP-binding pocket (14) are in bold.



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  		FIG. 2. Nucleotide sequence of MS11A ffs and the 4.5S RNA. The RNA sequence is shown below the DNA sequence, beginning at the first nucleotide following the presumed RNase P site. IVT from the T7 promoter on plasmid pBlu-ffsM13 begins at +1 (in bold). Oligonucleotides CGA76 and CGA77 were used for RT-PCR cloning as described in the text, with the restriction sites shown. Flanking sequences are from the vector pBluescript II SK- (Stratagene). CGA94 and CGA77 were used to produce the template for IVT. The domain IV region of the RNA shown to specifically interact with Ffh (4) is underlined.

RNA manipulations. Total low-molecular-weight RNA from N. gonorrhoeae strains MS11A and FA1090 was isolated with the RNA/DNA purification kit (Qiagen, Valencia, Calif.). cDNA was made from this RNA with murine leukemia virus reverse transcriptase and random primers (Applied Biosystems), and the resulting cDNA was PCR amplified with oligonucleotide primers CGA76 and CGA77 to produce ffs. Production of large quantities of 4.5S RNA was done by IVT with purified T7 RNA polymerase (Promega, Madison, Wis.) and a PCR-amplified PT7-ffs construct (with oligonucleotide primers CGA94 and CGA77; Fig. 2) as the template. For gel retardation and filter-binding assays, radioactively labeled 4.5S RNA was produced by the addition of [{alpha}-32P]ATP (Amersham Biosciences, Piscataway, N.J.) to the IVT reaction mixture. Following IVT, the DNA template was removed by DNase I treatment and the RNA was purified by extraction from 6% acrylamide-8 M urea gels. Purified RNA was quantitated spectrophotometrically.

Protein purification. PilA was purified as previously described (3). To produce large amounts of Ffh for purification, BL21{lambda}(DE3)/pETFfh-1 was grown in Luria broth at 30°C to mid-exponential phase (optical density at 600 nm, 0.4 to 0.8) and Ffh production was induced by the addition of 500 µM isopropyl-ß-D-thiogalactopyranoside (IPTG), followed by an additional 2 h of growth. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris [pH 7.5], 1 mM EDTA, 0.1 mM dithiothreitol) (2) containing 0.1 M KCl and a protease inhibitor cocktail (Bio-Rad Laboratories, Richmond, Calif.), and broken by sonication, and insoluble debris was removed by centrifugation. The soluble extracts were applied to a cation-exchange column (SP-Sepharose FastFlow; Pharmacia) and washed with multiple column volumes of buffer A, and proteins were eluted with a linear gradient of KCl (0.1 to 1 M) in buffer A with a BioLogic LP Low-Pressure System (Bio-Rad). Ffh eluted from this column at around 500 mM KCl. Ffh-containing fractions were pooled, adjusted to 2 M KCl, and chromatographed on Butyl Sepharose 4 FastFlow (Pharmacia). The column was washed with several column volumes of buffer A plus 2 M KCl and then with several column volumes of buffer A plus 1 M KCl, and finally proteins were eluted with a decreasing gradient of 1 to 0 M KCl. Recovery of all of the Ffh required additional washing of the column with buffer A (no KCl). The typical yield of Ffh (>=95% pure) from a liter of cells was 8 mg. Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, Ill.) or by the Bradford method (Bio-Rad), depending on the presence of reducing agents and detergents in the samples. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25) and stained with Coomassie blue. For amino-terminal sequence analysis of proteins, preparations separated by SDS-PAGE were electrophoretically transferred to polyvinylidene difluoride membranes and stained with Coomassie blue and appropriate bands were excised. Protein sequence analysis was performed on an ABI 475 protein sequencer (Applied Biosystems) by the research facility of the Shriners Hospital—Portland Unit.

GTPase assays. GTPase assays were done as previously described (3). Briefly, the reaction buffer contained 50 mM Tris-Cl (pH 8.0), 1 mM dithiothreitol, 0.1 mM EDTA, 10 mM MgCl2, and 10% glycerol. [{gamma}-32P]GTP (Amersham) was diluted in 1 mM GTP to 102 to 103 cpm/pmol. The total [GTP] in each reaction mixture was 100 µM unless otherwise indicated. Reactions were initiated by the addition of substrate (GTP) and carried out for 60 min at 37°C. The reactions were stopped, and free 32P was separated from [{gamma}-32P]GTP by organic extraction (35). Units were calculated as picomoles of GTP hydrolyzed per minute as a function of the protein concentration.

Gel retardation assays. Gel retardation assays to examine binding of Ffh and/or PilA to 4.5S RNA were done as previously described (2), with the following modifications. Binding reaction mixtures contained yeast tRNA at 5 µg/ml as a nonspecific competitor, and gels contained 7% polyacrylamide (29:1 acrylamide/bisacrylamide ratio). The electrophoresis buffer used (TBE) contained 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA, pH 8.0.

Filter-binding assays. Ffh binding to 4.5S RNA was measured by using a nitrocellulose filter binding assay (19). 32P-labeled 4.5S RNA (1 nM) was incubated with Ffh in buffer containing 50 mM HEPES (pH 7.5), 70 mM ammonium acetate, 30 mM potassium acetate, 1 mM dithiothreitol, 0.02% Triton X-100, 0.1% bovine serum albumin, and 0.01 mg of yeast tRNA per ml. Following 30 min of incubation at 25°C, reaction mixtures were filtered through nitrocellulose filters (0.45-µm pore size; Millipore), and unbound RNA was removed by washing twice with 0.5 ml of binding buffer. RNA retained by the filter bound was measured by liquid scintillation counting. Data were analyzed and plotted with KaleidaGraph 3.5 software (Synergy Software).

Nucleotide sequence accession number. The ffg nucleotide and ffh nucleotide and predicted protein sequences have been submitted to the GenBank database and assigned accession numbers AF482014 and AF482013, respectively.


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RESULTS
 
Cloning of ffh from N. gonorrhoeae strain MS11A. Because our previous studies of the gonococcal SRP were carried out with N. gonorrhoeae strain MS11A (50), we chose to clone ffh from this strain as well (see Materials and Methods). DNA sequences from three independent clones (pETFfh-1, pETFfh- 2, and pETFfh-4) were compared to each other and to the FA1090 ffh sequence (Roe et al., Gonococcal Genome Sequencing Project). All three clones differed from the FA1090 sequence at nucleotide position 1347 (A->G), which resulted in an amino acid change of isoleucine to methionine (Fig. 1). Since all three had the same change, it is likely that this A is the correct base for MS11A ffh. Interestingly, this region of the protein contains a large number of methionine residues that are thought to form a hydrophobic signal sequence-binding pocket lined with "methionine bristles" (6, 22), suggesting that a methionine at this position is not at all unreasonable. Analysis of the predicted sequence of the MS11A Ffh protein (shown in Fig. 1) shows that it consists of 456 amino acids with a molecular mass of 49.5 kDa and an isoelectric point (pI) of 9.0, values that are similar to those of E. coli Ffh, which are 49.8 kDa and 9.4, respectively. MS11A Ffh is 66% identical and 77% similar to E. coli Ffh, which is consistent with the observation that the SRP54 homologs are the most highly conserved of the SRP components (28).

Expression and purification of gonococcal Ffh. E. coli strain BL21{lambda}(DE3) (52), which contains the gene for T7 RNA polymerase under the control of the Lac repressor, was transformed with pETFfh-1, which contains ffh under the control of a T7 promoter. Transformants were analyzed for Ffh production following induction with IPTG, and SDS-PAGE analysis of samples showed a significant increase in a band at 50 kDa following IPTG induction, consistent with the predicted size of Ffh (Fig. 3). Conditions determined empirically to be optimal for maximum production and solubility of Ffh were used to produce large amounts of Ffh by a two-column purification scheme (see Materials and Methods). A Coomassie blue-stained SDS-PAGE gel of purified Ffh is shown in Fig. 3. One problem encountered in the purification of Ffh was an apparent proteolysis of the full-length protein during the purification process. A significant amount of a lower-molecular-mass protein (~45 kDa) increased with the time of storage of crude and partially purified fractions (Fig. 3). In order to determine the nature of this protein, it and the presumed full-length Ffh protein were eluted from SDS-PAGE gels and subjected to limited N-terminal sequencing. The sequencing results indicated that the 50-kDa protein was indeed the full-length gonococcal Ffh protein (first 15 residues determined; Fig. 1). The sequence of N. gonorrhoeae Ffh differs significantly from that of the E. coli protein, such that they are easily distinguished from one another, ruling out possible contamination with E. coli Ffh. N-terminal sequence analysis of the smaller protein showed that it was an apparent proteolytic product of the gonococcal Ffh missing the first 50 amino acid residues (Fig. 1). It is not clear by what process this proteolysis occurs or if it is physiologically significant; however, inclusion of fresh protease inhibitors in all buffers and rapid purification significantly reduced the level of this product.



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  		FIG. 3. SDS-PAGE analysis of Ffh purification. Samples were separated on a 12.5% polyacrylamide and stained with Coomassie blue. Plus and minus lanes contained total proteins from BL21{lambda}(DE3)/pETFfh-1 induced (+) or not induced (-) with 500 µM IPTG for 2 h. The Pure lane contained 1 µg of purified Ffh, and the question mark indicates the degradation product of Ffh that is missing the amino-terminal 50 amino acids (Fig. 1). Molecular mass standards are indicated on the left.

Enzymatic analysis of purified Ffh. Purified Ffh was analyzed for GTPase activity as described previously for PilA (3) and was found to hydrolyze GTP with Michaelis-Menten kinetics (Fig. 4). With the data from Fig. 4 and a derivative of the Michaelis-Menten equation (10), the maximum velocity of Ffh GTP hydrolysis was calculated to be 3,500 pmol min-1 mg-1, slightly greater than that determined for PilA (2,000 pmol min-1 mg-1) (3). The Michaelis-Menten constant of Ffh for GTP is 14 µM, which is also slightly greater than the value determined for PilA (~10 µM) (3), both of which are well below the presumed intracellular concentration of GTP (36).



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  		FIG. 4. Plot of Ffh GTPase activity as a function of GTP concentration. Reactions were carried out for 60 min with 10 pmol of Ffh, as indicated in Materials and Methods, and the substrate (GTP) concentration was varied from 10 µM to 1 mM. Activity was calculated as picomoles of Pi released per minute per milligram of protein. Assays were performed in triplicate, and the error bars represent standard deviations.

To examine the possibility of synergy in the GTPase activities of Ffh and PilA, which has been demonstrated in other SRP systems (30, 33, 43), GTPase assays were performed with equimolar amounts of PilA and Ffh (5 pmol) either separately or together. As shown in Table 1, the rate of hydrolysis in the presence of both enzymes is significantly greater than the sum of the two separately (1.52 > [0.40 + 0.46] pmol/min; P = 0.01 by Student's t test), indicating a modest amount of stimulation. It is not possible, however, to determine which protein(s) is being stimulated in this experiment. These experiments also did not include the 4.5S RNA, which has been reported to stimulate SRP GTP hydrolysis (30, 39); therefore, the gonococcal 4.5S RNA was next isolated.


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  		TABLE 1. Effect of RNA on GTP hydrolysis by gonococcal SRP GTPasesa

Cloning and expression of gonococcal 4.5S RNA. The SRPDB website includes the gonococcal 4.5S RNA (from strain FA1090, which is the strain that has been sequenced) in an alignment of SRP RNAs (17; http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html). This sequence was used to design oligonucleotide primers for cloning of N. gonorrhoeae MS11A ffs (see Materials and Methods). One clone, pBlu-ffsM13, was selected and used as a template for IVT to produce 4.5S RNA. An ethidium bromide-stained gel of a representative IVT experiment shows that an RNA band of 139 nucleotides (nt) was produced when templates contained ffs but not when they did not (Fig. 5). The 4.5S RNA prepared by this method was purified and used in GTPase assays of PilA and Ffh to examine the effects of the 4.5S RNA on the enzymatic activity of these proteins.



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  		FIG. 5. IVT of 4.5S RNA from N. gonorrhoeae ffs plasmids. pBlu-ffsF11, FA1090 ffs; pHTH1, pBluescript II with a random insert; pBlu-ffsM13, MS11A ffs; pBluescript II, vector control; RNA ladder, RNA molecular size standards. IVT reaction mixtures were run on a 6% polyacrylamide-8 M urea gel, and bands were stained with ethidium bromide. The UV-illuminated gel was photographed with a Bio-Rad GelDoc system.

Effect of 4.5S RNA on SRP GTP hydrolysis. GTPase assays were carried out as previously described (3), with 5 pmol of each of the three purified SRP components, Ffh, PilA, and 4.5S RNA, in various combinations. The results of these assays are shown in Table 1. Addition of 4.5S RNA to PilA resulted in a significant increase in GTP hydrolysis (about fivefold; P < 0.0001), whereas there was no apparent effect on Ffh activity. When all three components were present, the level of activity was higher than the activity observed with PilA and Ffh together in the absence of 4.5S RNA (2.50 > 1.52 pmol/min; P = 0.003). However, since the activity observed in the presence of all three components is similar to the sum of the activities of each protein in the presence of 4.5S RNA (2.00 + 0.32 {approx} 2.50 pmol/min), it is likely that the increase is due to stimulation of PilA GTP hydrolysis. The 4.5S RNA stimulation of PilA GTP hydrolysis was sequence specific, as demonstrated by control experiments in which yeast tRNA was used as a nonspecific RNA (Table 1). A similar lack of stimulation was observed when either of two additional small RNAs (the 45-nt guide RNA, gCYb-558, and a 126-nt fragment of its cognate mRNA, CYb [27]) were added (data not shown), supporting the sequence specificity of the RNA stimulation.

A possible explanation for the lack of stimulation of Ffh GTPase activity by 4.5S RNA was that the enzyme preparation was contaminated with the corresponding E. coli RNA. It has been suggested that, in vivo, Ffh is nearly always tightly bound to 4.5S RNA (53). In order to test this possibility, the Ffh and PilA preparations were examined for the presence of RNA by radiolabeling with polynucleotide kinase and [{gamma}-32P]ATP. These experiments utilized both purified PilA and Ffh and also samples that were extracted with phenol prior to the kinase reaction to remove the protein and free any RNA that might be present. Following the kinase reaction, samples were precipitated with trichloroacetic acid (TCA), which does not precipitate unincorporated ATP. No TCA-precipitable counts were detected in this experiment. To confirm this result, the TCA precipitates were run on a nondenaturing polyacrylamide gel that was then exposed to X-ray film for an extended time to detect any labeled RNA. The only radioactivity observed was a small amount of unincorporated [{gamma}-32P]ATP at the bottom of the gel and the control samples that were spiked with purified labeled 4.5S RNA (data not shown). These results indicate that the Ffh and PilA preparations are free of contaminating RNA.

Effect of DNA on SRP GTP hydrolysis. PilA exhibits anomalous sequence-specific DNA-binding activity (2). At the time the DNA-binding activity was identified, PilA was thought to be a transcriptional regulator that bound to promoter DNA to activate and/or repress transcription of the associated gene (54). When PilA was determined to be the gonococcal FtsY protein (1), however, it was not clear why the protein would bind DNA in a sequence-specific manner. To assess whether DNA binding affects SRP GTP hydrolysis, various DNAs were produced by PCR and added to GTPase assays of PilA and Ffh; the results of these experiments are shown in Table 2. PilA GTP hydrolysis was stimulated ~12-fold by one DNA molecule, PpilE, compared to the 5-fold stimulation observed with the 4.5S RNA (Table 1). This stimulation was sequence specific, as shown by the lack of stimulation with nonspecific DNA (sheared, sonicated herring sperm DNA) and ffs, the PCR product used for IVT production of gonococcal 4.5S RNA. This effect was also specific for double-stranded DNA, since treatment of the PCR product with mung bean nuclease (to degrade single-stranded DNA) had no effect on the ability of PpilE to stimulate PilA GTP hydrolysis (data not shown). The PpilE fragment used in this experiment is identical to the fragment used in our previous gel retardation assays, demonstrating the DNA-binding activity of PilA (2), suggesting that this observed DNA-binding activity is of physiological significance. None of the DNAs exhibited a significant effect on Ffh GTP hydrolysis, similar to what was observed when various RNAs were added to Ffh.


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  		TABLE 2. Effect of DNA on GTP hydrolysis by gonococcal SRP GTPasesa

Gel retardation analysis of 4.5S RNA by SRP proteins. A direct interaction between Ffh and 4.5S RNA has been demonstrated by biochemical techniques (41, 62), as well as in crystal structure studies (4). Likewise, interactions between FtsY and the Ffh-4.5S RNA complex have been demonstrated (19, 39). Nevertheless, a direct interaction between the SRP docking protein (FtsY) and 4.5S RNA (in the absence of Ffh) has not been reported. Indeed, in the E. coli system, such an interaction has been reported not to occur (19). In light of our observation that 4.5S RNA stimulates PilA (FtsY) GTPase activity (Table 1), purified SRP proteins were examined for RNA-binding activity by a gel retardation assay. This assay is based on the observation that the migration of nucleic acids through polyacrylamide (or agarose) gels can be altered when one or more proteins are bound to it (9). The migration is affected by the shape of the nucleic acid, as well as the charge-to-mass ratio of the proteins bound to the nucleic acid and can therefore either retard or accelerate the migration of the nucleic acid, although retardation is most frequently observed. Gel-purified, 32P-labeled 4.5S RNA was incubated with various amounts of PilA and Ffh alone or together and electrophoresed on nondenaturing polyacrylamide gels. A representative autoradiograph of such an experiment is shown in Fig. 6. Twenty picomoles of Ffh (lane 4) was sufficient to retard the mobility of all of the RNA in the reaction mixture (1.3 pmol), while 80 pmol of PilA (lane 7) was necessary to bind the same amount of RNA and some binding was observed with 20 pmol of PilA. Both complexes (Ffh-4.5S RNA and PilA-4.5S RNA) barely entered the gel, which could be a reflection of aggregation of the proteins on the RNA, since it is not possible in our assay to determine the stoichiometry of the components of the complex, only to demonstrate an interaction. In order to determine where the proteins were in the complexes, duplicate gels were stained with Coomassie blue to visualize the proteins (data not shown). Free PilA (in the absence of 4.5S RNA) migrated significantly into the gel but remained at the top of the gel when complexed with 4.5S RNA. Free Ffh barely entered the gel and was indistinguishable from Ffh complexed with 4.5S RNA. This is likely a reflection of the very different charges of the two proteins (PilA pI = 5.0, Ffh pI = 9.0) in the reaction and electrophoresis buffers (pH 8.0).



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  		FIG. 6. Gel retardation analysis of 4.5S RNA by PilA and/or Ffh. Assays were performed as described in Materials and Methods. Sixty nanograms of 32P-labeled 4.5S RNA was incubated with the proteins indicated for 30 min at room temperature, electrophoresed on a 7% polyacrylamide gel in TBE buffer, and exposed to X-ray film. Lanes: 1, no protein; 2, 2 pmol of Ffh; 3, 5 pmol of Ffh; 4, 20 pmol of Ffh; 5, 5 pmol of PilA; 6, 20 pmol of PilA; 7, 80 pmol of PilA; 8, 5 pmol of Ffh plus 5 pmol of PilA; 9, 5 pmol of Ffh plus 20 pmol of PilA.

Combinations of Ffh and PilA with the 4.5S RNA yielded novel results (Fig. 6). While the individual protein-RNA complexes were almost completely retarded and barely entered the gels (lanes 4, 6, and 7), a unique complex was observed when both proteins were present (lanes 8 and 9). When as little as 5 pmol each of Ffh and PilA was incubated with 1.3 pmol of 4.5S RNA, nearly 50% of the RNA formed a band that penetrated the gel significantly more than the individual protein-RNA complexes. The distinct mobility of the band formed with such small amounts of the two proteins suggests interaction among all three of the components in this complex.

Filter-binding analysis of Ffh binding to 4.5S RNA. To more accurately quantify the binding of 4.5S RNA by Ffh, nitrocellulose-binding assays were performed. In these assays, a very small amount of gel-purified, 32P-labeled 4.5S RNA (1 nM) was incubated with increasing amounts of Ffh protein in binding buffer. The binding reaction mixtures were then subjected to filtration and washing to remove unbound RNA. Bound RNA was quantified by scintillation counting, the data were plotted, and the results are shown in Fig. 7. The maximum RNA binding was typically 70 to 95% and occurred at >=0.75 µM Ffh. A binding constant (Kd) for the Ffh-4.5S RNA interaction was determined as the Ffh concentration required to bind 50% of the maximum RNA bound, which we determined to be 210 nM by this assay.



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  		FIG. 7. Nitrocellulose filter binding of Ffh to 4.5S RNA. 32P-labeled 4.5S RNA was incubated with various concentrations of Ffh, and binding was determined by filtration through nitrocellulose filters. The percentage of the total RNA bound is plotted as a function of the Ffh concentration. The values shown are averages of three determinations, and error bars indicate standard deviations.


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DISCUSSION
 
We have isolated the genes for and purified all three components of the SRP of N. gonorrhoeae strain MS11A. Analysis of Ffh and PilA showed that both proteins have significant GTPase activity and that the kinetics are similar. Notably, the interactions between these proteins and with the 4.5S RNA exhibit unique features compared to those of other characterized SRP systems. Interactions between Ffh and FtsY have been reported for other prokaryotic SRP systems (30, 32, 39, 41, 43, 46, 58), although the role of 4.5S RNA in this interaction is not clear since, in some of these cases, the RNA was always present. In light of this and the evidence of multiple roles for the 4.5S RNA in SRP-dependent protein targeting, such as in the Ffh-FtsY interaction and the binding of Ffh to signal sequences (4, 8, 19, 29, 32), we examined the contribution of the gonococcal 4.5S RNA to GTP hydrolysis by each of the SRP GTPases alone and together. In the absence of 4.5S RNA, we observed a modest (about twofold) stimulation of total GTPase activity by the combined proteins compared to the sum of the activities of PilA and Ffh alone (Table 1). The inclusion of 4.5S RNA had a small effect on the combined activity of the two proteins together but did not affect the activity of Ffh alone. In contrast, addition of 4.5S RNA to PilA resulted in significant stimulation of GTP hydrolysis (about fivefold; Table 1). This stimulation of PilA GTP hydrolysis by 4.5S RNA is different from what has been reported for the other prokaryotic systems, in which the interactions between FtsY and 4.5S RNA are dependent on Ffh (20, 24, 30, 43).

Our results are different from those reported by others who studied the E. coli SRP system and found that the 4.5S RNA does not interact directly with FtsY (PilA) in the absence of Ffh (19). To date, there has been no evidence that FtsY orthologs in any system interact with RNA of any kind. Our finding of a specific interaction between N. gonorrhoeae PilA (FtsY) and 4.5S RNA, on the basis of both GTPase assays and gel mobility shift studies, is the first example of such an interaction in an SRP system. Although Ffh GTP hydrolysis was not affected by 4.5S RNA, gel retardation and filter-binding assays did establish that these components interact and that Ffh binds 4.5S RNA slightly better than does PilA. Furthermore, our data suggest that all three of the SRP components interact. It is worth noting that all assays were done in the absence of GTP, which has been reported to be necessary for the interaction between Ffh and FtsY in E. coli (21), again demonstrating that the gonococcal SRP differs significantly from previously characterized prokaryotic systems. Addition of the nonhydrolyzable GTP analog GTP{gamma}S had no effect on the gel retardation assays (data not shown). Thus, at least in the Neisseria SRP system, there appears to be more interaction among the three components than has been previously observed in other systems. Whether or not this is truly unique to neisseriae remains to be seen.

Our results also provide some insight into the previously puzzling observation that PilA binds to pilE promoter DNA in a sequence-specific manner (2). We now know that PilA, rather than functioning as a transcriptional regulator of gonococcal pilin biosynthesis, is part of the SRP protein targeting pathway (1). Here, our results show that PilA GTP hydrolysis is stimulated by DNA, also in a sequence-specific manner. Thus, both SRP RNA and the promoter DNA of a gene encoding an exported protein specifically stimulate the GTPase activity of the gonococcal SRP docking protein, an activity that is required for its function in protein targeting (1, 24, 55). These observations suggest that there might be a role for both DNA and RNA in SRP-dependent protein targeting.

In a current model of prokaryotic SRP function, ribosomes that are in the process of translating integral CM proteins form a complex with the SRP RNP (Ffh plus 4.5S RNA) via binding of the hydrophobic signal sequence of the nascent polypeptide, are targeted to the membrane-associated SRP receptor, FtsY, where the nascent protein is inserted into the SecYEG translocon and translocation occurs cotranslationally. Our observation that specific RNA (4.5S) and DNA (PpilE) sequences interact with and stimulate the GTPase activity of the gonococcal SRP receptor, PilA (FtsY), coupled with the well-known fact that prokaryotic transcription and translation are coupled, leads us to propose an addition to the current model of SRP targeting in which DNA plays a role. In this scenario, the transcription, translation, and translocation complexes are all present and in close proximity to one another, allowing interaction between DNA and PilA and thus compartmentalizing the expression and translocation of a CM protein. There are two possibilities for a role for DNA in the targeting process. First, a signal for targeting of the transcription-translation-SRP complex to the SRP receptor at the membrane might reside in a DNA sequence, perhaps a sequence in the promoter region of the gene encoding the respective membrane protein. Indeed, we have preliminary evidence that suggests that a DNA sequence specifically bound by PilA, PpilE, is the promoter region of a gene that encodes a protein, gonococcal pilin, that appears to confer an SRP-dependent phenotype when expressed in E. coli (data not shown). An alternative explanation, also consistent with our data, is that interaction between DNA and the SRP receptor is necessary for the stimulation of GTP hydrolysis, which is required for transfer of the translation complex to the translocon, and subsequent translocation of the protein. In either case, an interaction between 4.5S RNA and the SRP receptor could also occur and result in stimulation of GTP hydrolysis. Additional experiments to more closely examine the role of DNA binding in SRP function are necessary to test these possibilities.


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ACKNOWLEDGMENTS
 
This work was supported by start-up funds to C.G.A. from the Colleges of Human and Osteopathic Medicine at Michigan State University.

We thank Jay Gambee of the Portland Shriners Hospital for protein sequence analysis and the Oregon Health and Science University Department of Molecular Microbiology and Immunology Core Facility for DNA sequence analysis. We are also grateful to Maggie So for support in the early stages of this project and to Bob Hausinger for critical comments on the manuscript. We also thank Donna Koslowsky for providing the T. brucei RNA controls used in the PilA GTPase assays.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-1101. Phone: (517) 355-6463, ext. 1573. Fax: (517) 353-8957. E-mail: arvidso3{at}msu.edu. Back


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Journal of Bacteriology, February 2003, p. 801-808, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.801-808.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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