Tài liệu Báo cáo khoa học: Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails ppt

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails Marc Folichon, Fre ´ de ´ ric Allemand, Philippe Re ´ gnier and Eliane Hajnsdorf UPR CNRS 9073, conventionne ´ e avec l’Universite ´ Paris 7 – Denis Diderot, Institut de Biologie Physico-Chimique, Paris, France Host factor I (Hfq) is an abundant protein of Escherichia coli, which was first identified as a host factor required for the replication of Qb bacterio- phage [1]. It has then been established that disrup- tion of the hfq gene causes pronounced pleiotropic phenotypes in uninfected E. coli [2] and that in other bacteria Hfq permits the adaptation to multiple envi- ronmental stresses [3,4]. There is now an accumulation of data that shows Hfq is an RNA-binding protein that is associated with RNA replication, translation and stabilization [5–8]. In the case of phage Qb replication, Hfq acts directly by bringing into close proximity the 3¢ terminal and internal regions of the genomic RNA [9]. Hfq was also reported to weaken base-pairing in stem loops of OxyS sRNA, to mask the ribosome binding site of ompA mRNA [10], to protect several RNAs from deg- radation by ribonucleases [11,12] and to assist many sRNAs in their activity [5,13–16]. In this latter case, it was shown that Hfq facilitates base-pairing between small regulatory RNAs and their mRNA target [17,18]. These properties and its capacity to rescue a splicing defective intron from a ‘folding trap’ led to the proposal that Hfq is an RNA chaperone acting at many different steps of RNA metabolism [15,19–21]. In addition, the presence of a characteristic Sm fold and its organization in a crown-shaped homo-hexamer proved that Hfq belongs to the Sm-like protein family [18,22–24] whose members are involved in many RNA–RNA and RNA–protein transactions. Finally, Hfq was also recently reported to interact with ribo- somal protein S1 and RNA polymerase, to exhibit an Keywords E. coli; Hfq protein; poly(A)-polymerase I; polynucleotide phosphorylase; RNA–protein interaction Correspondence E. Hajnsdorf, UPR CNRS 9073, conventionne ´ e avec l’Universite ´ Paris 7 – Denis Diderot, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France Fax: +33 1 58 41 50 20 Tel: +33 1 58 41 51 26 E-mail: Eliane.Hajnsdorf@ibpc.fr (Received 16 September 2004, revised 15 November 2004, accepted 16 November 2004) doi:10.1111/j.1742-4658.2004.04485.x The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stabil- ity, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3¢ end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq–RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A). Abbreviations Hfq, host factor I; PAP I, poly(A)polymerase; PNPase, polynucleotide phosphorylase. 454 FEBS Journal 272 (2005) 454–463 ª 2004 FEBS ATPase activity and to affect polyadenylation of bac- terial RNAs [25–27]. Polyadenylation is ubiquitous in all organisms but has opposite effects on mRNA stability in prokaryotes and eukaryotes. These two opposed functions of poly- adenylation appear to be represented in the mitochon- dria of different organisms [28]. In the chloroplast, however, there is no equivalent of poly(A)polymerase and the same enzyme, polynucleotide phosphorylase, polyadenylates and degrades the RNA molecule [29]. In some cases, factors have been identified which stimulate the activity of poly(A)polymerase, rendering the reaction processive; in E. coli, this function is assured by Hfq. We have demonstrated that Hfq stimulates poly(A) elongation catalyzed by poly(A)po- lymerase (PAP I) and that tails are synthesized very rapidly once RNA have acquired a tail of 20–35 resi- dues [26]. In vivo, Hfq favors the appearance of long poly(A) tails, it increases the fraction of polyadenylated rpsO mRNAs and it modifies the repartition of the poly(A) tails at the 3¢ ends of RNA species resulting from endo- and exonucleolytic processing [26,27]. However, we do not know how Hfq affects substrate recognition and poly(A) elongation by PAP I. In support of the idea that Hfq binding to the 3¢ end of RNA facilitates PAP I activity, we show here that Hfq does not stimulate the PAP I mediated elon- gation of poly(C) tails for which it has no affinity. Moreover, we find that RNA harboring 5¢ monophos- phorylated extremities, which favor RNase E process- ing and poly(A) dependent exonucleolytic degradation, are not preferentially bound by Hfq. This suggests that Hfq is not involved in the 5¢ end dependent activation of RNA decay. Results Failure to stimulate poly(C) synthesis is correlated with inefficient binding of Hfq to poly(C) tailed RNA We reported previously that Hfq stimulates elongation of poly(A) tails, but it is not known whether this reflects a modification of the RNA substrate due to the formation of an Hfq–RNA complex or to a direct interaction of Hfq with PAP I. To better understand the mechanism of activation, we have investigated whether stimulation of PAP I activity is correlated with the affinity of Hfq for the 3¢ end of RNA. We used an RNA fragment corresponding to the 3¢ end of the rpsO transcript, which was shown to be polyaden- ylated both in vivo and in vitro [26,30,31]. For that purpose, we have first examined Hfq binding to 3¢rpsO RNA fragments harboring different homopolymeric tails of 18 nucleotides or a stretch of 18 encoded nucleotides lying downstream of the transcription ter- minator of the polycistronic rpsO-pnp mRNA (5¢-AA GCUGACGGCAGCAAUU). As seen in Fig. 1A, we find that Hfq binds much more efficiently RNAs that harbor poly(A) or poly(U) tails than RNA harboring poly(G) or poly(C). These results are in agreement with previous data obtained with homopolymers [22]. Comparison with tail-less RNA suggests that poly(G), poly(C) tails and the natural stretch of 18 nucleotides encoded downstream of the rpsO transcription termi- nator are either not, or only inefficiently, bound by Hfq. In order to determine which RNA substrate is more efficiently bound by Hfq, we performed competi- tion experiments using poly(A) and poly(U) tailed 3¢rpsO RNA. The gel-shift experiments of Fig. 1B clearly show that Hfq exhibits a preference for poly- adenylated molecules over RNA tailed with a poly(U) sequence. It must be mentioned here that the intracel- lular Hfq concentration (about 10 lm [32]) is much higher that the concentration used in the experiment. We next examined whether Hfq stimulates synthesis of a homopolymeric tail that it does not recognize. To avoid synthesis of a poly(A) tail, which would create an Hfq binding site at the 3¢ end of the RNA, we investigated whether Hfq stimulates elongation of poly(C) tails for which it exhibits a very low affinity. This approach was valudated by previous results indi- cating that, in spite of its high preference for ATP, PAP I can also achieve CTP elongation by approxima- tively 500 nucleotides [33]. Accordingly, we found that PAP I slowly polymerises C residues at the 3¢ end of the 3¢rpsO substrate in our experimental conditions [34,35] (Fig. 2A). Interestingly, Fig. 2B clearly shows that Hfq does not affect synthesis of poly(C) tails by PAP I. Hfq (160 nm) does not inhibit poly(C) synthesis but at this Hfq concentration and at higher concentra- tions (data not shown) degradation of the RNA sub- strate can be detected; this faint degradation is also seen in lane 7 of the same figure. Some nonadenylated substrate remains in the presence of Hfq (Fig. 2B) but also in the absence of Hfq (Fig. 2A, lane ATP). These small amounts of nonadenylated RNA fragment are not constant, nor proportional to Hfq concentration. These data demonstrate that Hfq does not stimulate elongation of an RNA molecule that it does not bind, and are consistent with the idea that stimulation of polyadenylation reflects the affinity of Hfq for the RNA rather than its interaction with PAP I. Alternat- ively, it is also possible that PAP I stimulation of poly(A) synthesis is correlated with the ATPase activ- ity of Hfq [25]. M. Folichon et al. Hfq binding to RNA stimulates elongation by PAP I FEBS Journal 272 (2005) 454–463 ª 2004 FEBS 455 The position of the AU rich region at the 5¢ end is not critical for Hfq binding The idea that stimulation of poly(A) synthesis is cor- related with the association of Hfq to the 3¢ end of RNA prompted us to investigate whether 3¢ terminal AU rich regions are preferentially bound by Hfq. If true, this may indicate that a function of Hfq in vivo could be to favor polyadenylation of RNAs harbor- ing such 3¢ elements. It is already known that Hfq binds the OxyS109 sRNA as well as mRNA frag- ments such as 3¢rpsO and ompA105. Moreover, it has been shown that appending a 3¢ terminal poly(A) or a single stranded stretch of nucleotides to these mRNA fragments (thus giving rise to 3¢rpsO-A 18 and ompA117, respectively) strongly enhances Hfq affinity (Fig. 3) [10,12]. We performed a series of gel-shift experiments in order to generate a hierarchy of Hfq binding efficiency to these different RNAs harboring internal or 3¢ terminal presumptive binding sites. Interestingly, Fig. 3 shows that Hfq exhibits roughly the same affinity for the 3¢rpsO-A 18 RNA harboring a3¢ terminal tail and the OxyS109 RNA that was proposed to be bound by Hfq at an internal site framed by two stable hairpins [13]. This suggested that Hfq binding is independent of the location of the AU rich region in the RNA. This conclusion has been verified by comparing the affinity of Hfq for 3¢rpsO RNA harboring a stretch of 18 A residues appended either at the 3¢ end (3¢rpsO-A 18 ) or between the two secondary structures. This last RNA is bound by Hfq with an affinity similar to that of the RNA harboring a 3¢ terminal poly(A) tail. As shown in Fig. 4, two slowly migrating complexes were detected when the A 18 sequence is internal, while three complexes were detected with the A 18 sequence at the A B Fig. 1. Relative affinity of Hfq for 3¢ A 18 ,3¢ U 18 ,3¢ C 18 ,3¢ G 18 or 3¢ N 18 tailed rpsO mRNAs. 5¢ end labeled RNA and RNA 3¢ end tailed with various homopolymer and heteropolymer sequences were mixed with Hfq and analyzed on native gel. N 18 represents the 18 nucleotide sequence 5¢-AAGCUGACGGCAGCAAUU. (A) Hfq binding to various RNA substrates. The different 5¢ end labeled RNAs (20 p M) indicated at the bottom of the graph were mixed with 20 p M Hfq-His6 and formation of complexes was analyzed by gel-shift assay. The quantification of the gel is shown. (B) Competition assay. The 5¢ labeled RNA indicated at the bottom of the autoradiograph (20 p M) was incubated without (–) (lane 1) and with 10 p M Hfq-His6 (+) (lanes 2–7) and increasing amounts of the competitor RNA indicated at the top of the autoradio- graph; 20 p M (lane 3), 40 pM (lane 4), 100 pM (lane 5), 200 pM (lane 6) and 400 pM (lane 7). Hfq binding to RNA stimulates elongation by PAP I M. Folichon et al. 456 FEBS Journal 272 (2005) 454–463 ª 2004 FEBS AB Fig. 2. Hfq stimulates addition of A residues by PAP I but not of C residues. (A) Addition of various residues by PAP I. 5¢ end labeled p-3¢rpsO mRNA was incubated for 15 min at 37 °C, without PAP I (–) and with the NTP indicated at the top of the panel. Samples were analyzed on a sequencing 6% acrylamide gel along with radioactive DNA size markers. (B) Hfq stimulation of PAP I elongation in the presence of ATP or CTP. The 5¢ end labeled p-3¢rpsO mRNA was mixed without (lanes 1 & 8) and with increasing Hfq concentrations; 5 n M (lanes 2 & 9), 10 n M (lanes 3 & 10), 20 nM (lanes 4 & 11), 40 n M (lanes 5 & 12), 80 nM (lanes 6 & 13) and 160 n M (lanes 7 & 14). Incubation was conducted in the presence of PAP I for 15 min at 37 °C, in the presence of ATP (lanes 1–7) or CTP (lanes 8–14). Fig. 3. Comparison of Hfq binding to various RNA. (A) Predicted structures of the RNA used in the gel retardation experiments. The RNA sequences were folded with MFOLD (http://www.bioinfo.rpi.edu/applications/mfold) and their size is indicated in brackets (nt, nucleotides). Num- bers on the secondary structures indicate the length of the single stranded regions. Black rectangles locate Hfq binding sites determined by enzymatic and chemical probing and by mutational analysis. Grey rectangle locates the Hfq binding site determined by gel retardation assay. Grey curves locate AU rich sequences. 5¢ end labeled RNA (20 p M) were incubated in the absence and in the presence of 20 pM and 200 pM Hfq- His6 protein. Complexes were separated from unbound RNA substrates as described in Fig. 1. (B) PhosphoImager analysis of the results. The 5¢ end labeled RNA indicated at the bottom of the figure was incubated with 20 p M (open columns) and 200 pM (gray columns) of Hfq protein. M. Folichon et al. Hfq binding to RNA stimulates elongation by PAP I FEBS Journal 272 (2005) 454–463 ª 2004 FEBS 457 3¢ end. We conclude that the presence of secondary structures at both extremities of the potential Hfq binding site may limit the number of Hfq molecules susceptible to interact with the RNA, but that the position of the 18 A residues relative to secondary structures does not modify the affinity of Hfq for RNA. These data prompted us to examine whether any RNA fragment containing AU rich single-stranded sequences and secondary structures may be bound by Hfq. For that purpose, we synthesized an RNA frag- ment which corresponds to the 5¢ part of rpsO mRNA (5¢rpsO). Examination of its secondary structure reveals a bulge loop containing a UUUUAAAAUGU sequence and an 11 nucleotide long linker containing 6 Us and 3 As [36]. It is interesting that this RNA frag- ment, which is not known to interact with Hfq in the cell, is bound by Hfq as efficiently as the ompA117 mRNA fragment which contains an Hfq binding site implicated in translation and stability of this mRNA (Fig. 3). These data may indicate either that structural determinants different from the AU rich stretch of nucleotides are required for efficient Hfq binding in vivo or that Hfq interacts with the 5¢rpsO RNA fragment which contains the translational operator of the rpsO messenger [37]. The phosphate number at the 5¢ end of the RNA does not affect Hfq binding We have also examined whether the phosphorylation status of the 5 ¢ end of the RNA fragment, which was shown to affect poly(A) synthesis of RNA I, a highly folded unstranslated regulatory RNA [38], poly(A) dependent decay [39,40] as well as RNase E and RNase G processing [41–43] also modulates Hfq binding. To address this question, two forms of the (3¢rpsO-A 18 ) RNA substrate differing by the phosphorylation state of their 5¢ extremity were synthesized. As usual, the tri- phosphorylated substrate was prepared by in vitro tran- scription in the presence of all four nucleoside triphosphates and [ 32 P]UTP[aP]. The reaction mixture for the in vitro synthesis of the monophosphorylated substrate contained [ 32 P]UTP[aP] as a tracer and a large excess of guanosine over GTP, generating non- phosphorylated 5¢ ends which were then selectively labeled by T4 polynucleotide kinase in the presence of [ 32 P]ATP[a ˜ P]. As shown in Fig. 5A, Hfq binds both mono- and tri-phosphorylated RNA substrates with the same efficiency. In both cases, four complexes were observed by increasing Hfq concentrations. These data agree with previous identifications of two Hfq binding sites on the mono-phosphorylated 3¢rpsO-A 18 [12]. We cannot determine if higher complexes result from Hfq binding to other unidentified sites or whether protein– protein interactions account for the appearance of these complexes. We concluded that, in contrast to the many processes quoted above, Hfq binding does not depend on the phosphorylation status of the 5¢ extremity (Fig. 5A). We also examined whether polyadenylation effic- iency of an mRNA fragment is influenced by the phos- phorylation state of its 5¢ extremity as previously reported for RNA I of colE1 plasmid. As shown in Fig. 5B, PAP I is more active on the mono- than on the tri-phosphate RNA substrate; poly(A) tailed p-3¢rpsO are, on average, 150 nucleotides longer after 30 min than poly(A) tailed ppp-3¢rpsO. Our result extends to mRNA the previous data obtained with small regulatory RNA (Fig. 5B). Hfq inhibits poly(A) synthesis by polynucleotide phosphorylase Because Hfq binds to the 3¢ end of RNA harboring a single stranded stretch of nucleotides, we have investigated whether Hfq also affects the activity of Fig. 4. Relative affinity of Hfq for rpsO mRNA tailed at its 3¢ end with 18 A residues or containing an internal A 18 sequence. The 5¢ labeled RNA indicated at the bottom of the autoradiograph (10 p M) was incubated without (–) and with (+) 200 p M Hfq protein. Com- plex formation was analyzed on native gel. The arrows indicate the position of the A 18 sequence. Hfq binding to RNA stimulates elongation by PAP I M. Folichon et al. 458 FEBS Journal 272 (2005) 454–463 ª 2004 FEBS polynucleotide phosphorylase (PNPase). It has previ- ously been proposed that PNPase also synthesises tails in vivo [44]. For that purpose we have looked at the elongation of 5¢ labeled polyadenylated 3¢rpsO RNA by purified PNPase in the presence of ADP. Data shown in Fig. 6 suggest that Hfq impairs access of PNPase to the substrate but does not affect the elongation of the tails. Indeed we observed that the addition of Hfq delays utilization of the substrate (this effect increases with the amount of Hfq) but that it does not affect appearance of the long tails. This can be explained easily by assuming that Hfq does not impair the rate of processive elongation of RNA by the PNPase molecule, which remains bound to the polynucleotide. One can propose that Hfq may mask the 3¢ end of RNA (namely the 18 A residues at the 3¢ end of the substrate) and therefore prevent recycling of PNPase from a molecule that has been elongated to a new molecule of primer. In contrast, once the RNA–PNPase complex is formed and pro- cessive elongation engaged, Hfq can probably not affect the rate of polymerization. These data are con- sistent with the idea that Hfq forms a complex with RNA which, in this case, has an inhibitory effect on binding of poly(A) by PNPase. In contrast, an inter- action between Hfq and PNPase would be expected to inhibit both initiation and elongation of the tail. Discussion In this report, we show a correlation between Hfq binding to the 3¢ end of an RNA molecule and its abil- ity to stimulate PAP I which suggests that the Hfq– RNA interaction facilitates RNA recognition by PAP I. Moreover, we also demonstrate that Hfq binds very efficiently to RNA harboring stretches of poly(A) and poly(U) and that the location of this structural feature in the molecule (i.e. whether it is internal or 3¢ terminal) does not affect the affinity of Hfq. Hfq bind- ing does not depend upon the phosphorylation status of the 5¢ end. Finally, we show that formation of a Hfq–poly(A) complex which activates poly(A) synthe- sis by PAP I, inhibits poly(A) synthesis by PNPase, suggesting that the two enzymes interact differently with 3¢ extremities. Our data suggest that PAP I preferentially elongates RNA harboring poly(A) tails bound by Hfq. It is poss- ible that structural modifications resulting from Hfq binding facilitate recognition of the 3¢ end or its adeny- lation by PAP I [12]. It is worth recalling here, that similarly, an interaction between the 5¢ extremity of an RNA with its 3¢ end was proposed to explain why 5¢ monophosphorylated RNAs are more efficently adeny- lated by PAP I than those harboring a triphosphoryl- ated 5¢ end [38]. AB Fig. 5. A monophosphate 5¢ end stimulates polyadenylation but not Hfq binding. (A) Hfq binding to mono- and tri-phosphorylated RNA sub- strates. 5¢ end labeled p-3¢rpsO mRNA and of uniformly labeled ppp-3¢rpsO mRNA (50 p M) were incubated without (lanes 1 & 5) and with increasing amounts of Hfq-His6; 100 p M (lanes 2 & 6), 500 pM (lanes 3 & 7) and 2.5 nM (lanes 4 & 8). (B) Polyadenylation of RNA substrates harboring a 5¢ mono- or a 5¢ tri-phosphorylated extremity. Uniformly labeled ppp-3¢rpsO mRNA and 5¢ end labeled p-3¢rpsO mRNA were incu- bated with 1.2 pmol PAP I. Samples were taken at the times indicated at the bottom of the figure. The two time course experiments were analyzed by electrophoresis on the same gel along with radioactive DNA markers. For convenience, two exposures of the same gel are shown. M. Folichon et al. Hfq binding to RNA stimulates elongation by PAP I FEBS Journal 272 (2005) 454–463 ª 2004 FEBS 459 Interestingly, the hierarchy and the relative affinities of Hfq to homopolymeric tails [poly(A) > poly(U) >> poly(C) > poly(G)] is similar to that previously described in the case of the Staphylococcus aureus pro- tein for oligonucleotides [22], suggesting that tails of long RNA also bind the sites surronding the central hole of the Hfq hexamer. However, the much higher stability of complexes formed with long RNA com- pared to that of the Hfq-oligos [22,45] indicates that Hfq establishes additional contacts with RNA that reinforce the strength of the association [46]. These sites may be similar to those described at the surface of an archael Sm-like protein [47]. Our previous data showing that Hfq binds long poly(A) sequences [poly(A 115 )] as efficiently as poly- adenylated molecules suggests that the length of the RNA rather than secondary structures could account for the formation of stable complexes [12]. One could postulate that secondary structures stimulate Hfq bind- ing when they prevent intramolecular annealing of RNA sequences with AU rich regions preferentially recognized by Hfq. We have shown here and in our previous study [12] that the Hfq–poly(A) complex, which facilitates elonga- tion of the RNA by PAP I, prevents its recognition by PNPase which inhibits both degradation and elongation of the RNA by this latter enzyme. These data imply that different structural features of RNA are recognized by the two enzymes. In the case of PNPase, Hfq may mask the secondary site that was proposed to facilitate RNA recognition and processivity of the reaction [48]. Failure of Hfq to impair the processivity of the reaction (Fig. 6) suggests that it does not compete with PNPase already engaged in processive elongation of the RNA. These data also suggest that PAP I does not interact with single-stranded RNA upstream of the 3¢ end of the molecule which is presumably masked by Hfq. In addi- tion, one can also speculate that the synthesis of long tails, which was attributed to PNPase, is presumably strongly inhibited by Hfq in vivo. Finally, although our data suggest that structural modification of RNA due to Hfq binding, account for stimulation of PAP I medi- ated poly(A) synthesis, the recent demonstration that Hfq interacts with proteins such as ribosomal protein S1 and RNA polymerase implies that Hfq also establi- shes protein–protein interactions that may affect physiological functions of its protein partner. Materials and methods Protein purification Hfq-His6 was overproduced and purified as described [12]. Hfq without the His-tag was produced from the pET-Hfq plasmid, which was constructed as follows. A PCR fragment was generated using reverse and forward primers with the sequences 5¢-GGGAATTCCATATGGCTAAGGGGCAA TC and 5¢-AGGATCGCTGGATCCCCGTGTAAAAAA AC, respectively, with pTX367 plasmid [8], creating an NdeI site that included the translation initiation codon and a BamHI site downstream of the terminator. The PCR frag- ment digested by NdeI and BamHI was inserted into pUC18 vector that had been digested with the same enzymes. The Hfq containing DNA fragment was excised with the same enzymes and ligated into the corresponding sites of pET11c and then transformed into the E. coli strain BL21(DE3). Cells in 10 mL lysis buffer [100 mm Tris ⁄ HCl (pH 7.5), 800 mm LiCl, 150 mm MgCl 2 ,1mm dithiothreitol, antipro- tease cocktail complete, mini-EDTA free (Mannheim GmbH, Germany)] were disrupted using a French press (twice at 85 MPa at 4 °C). The lysate was incubated on ice for 20 min with 1 lgÆmL )1 Dnase I. The cell debris was removed by centrifugation at 9000 g for 30 min, the resul- tant clear lysate supernatant was precipitated by ammonium sulfate (70% saturation). The precipitate was pelleted and resuspended in 10 mL Binding Buffer [50 mm Tris ⁄ HCl (pH 8), 50 mm NaCl, 10% glycerol, 1 mm dithiothreitol] and then dialyzed overnight against the same buffer at 4 °C. The suspension was injected onto a Q FF column (16 ⁄ 20 Amersham Biosciences Europe GmbH, Saclay, France). Fractions eluting at 200 mm NaCl were pooled and dialyzed Fig. 6. Hfq inhibits the addition of A residues by PNPase. Elonga- tion of 5¢ labeled polyadenylated 3¢rpsO mRNA by PNPase was per- formed in the absence and in the presence of 10 n M and 100 nM Hfq-His6 as described in Materials and methods. Aliquots were removed at 0.5, 2, 3 and 5 min and analyzed on an acrylamide sequencing gel. Hfq binding to RNA stimulates elongation by PAP I M. Folichon et al. 460 FEBS Journal 272 (2005) 454–463 ª 2004 FEBS overnight at room temperature in Binding Buffer. The solu- tion was applied onto a Hi-trap Heparin column (Pharma- cia) and eluted with a linear NaCl gradient between 500 and 600 mm NaCl. The pooled fractions were dialyzed overnight at 4 °C into 50 mm Tris ⁄ HCl (pH 7.5), 50 mm NH 4 Cl, 5% glycerol, 1 mm EDTA (Buffer A). The sample was loaded onto a poly(A)-column [poly(A) Sepharose 4B Pharmacia] equilibrated with Buffer A at 4 °C [49]. The run through was collected and reloaded. The column was washed at 4 °C with 50 mm Tris ⁄ HCl (pH 7.5), 1 m NH 4 Cl, 5% glycerol, 1mm EDTA (Buffer B) and then transfered to room tem- perature. Hfq was eluted with Buffer B plus 8 m urea and dialyzed at 4 °C into Buffer A with 0.5 mm dithiothreitol. Purified Hfq was stored at 4 °C in the same buffer contain- ing 0.1% Triton. Hfq and Hfq-His6 were used interchange- ably [12] and their concentrations are expressed as monomer concentration. PAP I was overproduced from plasmid pPAP and purified as in [50]. RNA preparation and labeling Templates for synthesis of the different RNAs were obtained by PCR amplification of the first 125 nucleotides of the rpsO mRNA (5¢rpsO), the last 97 nucleotides of the same transcript (3¢rpsO), part of the 5¢ UTR of ompA transcript (ompA117 and ompA105) and the OxyS sequence (oxyS109) using the primers described in Table 1. The templates used to synthesize 3¢rpsO-RNA with 3¢ tails of 18 A, 18 C, 18 G, 18 U or 18 N (5¢-AA GCUGACGGCAGCAAUU) residues were transcribed using the corresponding reverse primers. The template used to synthesize the 3¢rpsO RNA containing an inser- tion of 18 A residues between the two hairpins was obtained after two PCRs using first the forward rpsO internal primer and the reverse 3¢rpsO primer, and then, the first PCR product and the forward 3¢rpsO primer. The PCR products were purified on an agarose gel after each step. Transcription reactions were carried out as in [12] using [ 32 P]UTP[aP] as tracer, yielding uniformly labeled RNA. When needed, 20 mm guanosine was also added to allow further direct 5¢ labeling. Radiolabeled RNAs were gel puri- fied and resuspended in water. RNA concentrations were monitored by counting out the radioactivity. Labelling of the 5¢ end was performed with [ 32 P]ATP[a ˜ P] and T4 poly- nucleotide kinase, labeled RNAs were separated from un- incorporated nucleotides through a ProbQuant G-50 Microcolumn (Amersham Biosciences). Electrophoretic mobility shift assays Hfq protein was incubated with 5¢ [ 32 P]RNA in 20 lL buffer containing 10 mm Tris⁄ HCl (pH 8), 1 mm EDTA, 80 mm NaCl, 1% glycerol (v ⁄ v). Reactions were incubated at 37 °C for 30 min and complexes were resolved by electrophoresis through native polyacrylamide gel [13]. A PhosphoImager and the imagequant software (Amersham Biosciences Europe) were used to view the gel and to quantify results. Polyadenylation in vitro Polyadenylation by PAP I was conducted as in [26] using 5¢ end labeled RNA or uniformly labeled 3¢rpsO RNA with purified PAP I (77 fmol). Samples were analyzed on dena- turing 6% polyacrylamide gel. Poly(A) tail synthesis by PNPase was conducted using 5¢ end labeled 3¢rpsO-A 18 RNA (2 pmol) in 50 lL; 50 mm Tris ⁄ HCl (pH 8), 5 mm MgCl 2 ,50mm NaCl, 0.1 mm dithiothreitol, 0.5 mgÆmL )1 tRNA, 0.5 mm ADP with 2 pmol PNPase. Acknowledgments We are grateful to J. Plumbridge for careful critical reading of the manuscript. We thank C. Portier for providing PNPase, A. J. Carpousis, T. Elliott and Table 1. PCR primers. Itallics correspond to RNA polymerase sequences of the T7 or T3 phages (T3 phage indicated by *), respectively. Primer name Sequence Forward ompA* 5¢-TAATTAACCCTCACTAAAGGGGTGCTCGGCATAAG Reverse ompA105 5¢-GCCATGAATATCTCCAACGAG Reverse ompA117 5¢-CATCCAAAATACGCCATGAATATC Forward 5¢rpsO 5¢-TAATACGACTCACTATAGGGGCCGCTTAACGTCGCG Reverse 5¢rpsO 5¢-GCTTCAGTACTTAGAGAC Forward 3¢rpsO 5¢-TAATACGACTCACTATAGGGAGACGTAGCACGTTACACC Reverse 3¢rpsO 5¢-GAAAAAAGGGGCCACTCAGG Reverse 3¢rpsO-(T)18 5¢-T(18)GAAAAAAGGGGCCACTCAGG Forward rpsO internal 5¢GCGTCGCTAATTCTTGCGAGA18TTTCAGAAAAGGGCTG Reverse 3¢rpsO-(C)18 5¢-C(18)GAAAAAAGGGGCCACTCAGG Reverse 3¢rpsO-(G)18 5¢-G(18)GAAAAAAGGGGCCACTCAGG Reverse 3¢rpsO-(N)18 5¢-GAATTGCTGCCGTCAGCTTGA Forward oxyS109* 5¢-TAATTAACCCTCACTAAAGGGAAACGGAGCGGCACCTCTT Reverse oxyS109 5¢-GCGGATCCTGGACCGCAAAAG M. Folichon et al. Hfq binding to RNA stimulates elongation by PAP I FEBS Journal 272 (2005) 454–463 ª 2004 FEBS 461 M. Winkler for the gifts of pPAP, pTE607 and pTX367, respectively. This work was supported by Universite ´ Paris 7 – Denis Diderot (plan quadriennal), the Center National de la Recherche Scientifique (UPR 9073), the Programme de Recherche Fondamen- tale en Microbiologie et Maladies Infectieuses et Paras- itaires of the Ministe ` re de l’Education Nationale de la Recherche et de la Technologie. M. F. is recipient of a grant from M. N. R. T. References 1 Franze de Fernandez MT, Hayward WS & August JT (1972) Bacterial proteins required for replication of phage Qb ribonucleic acid. J Biol Chem 247, 821–824. 2 Muffler A, Fischer D & Hengge-Aronis R (1996) The RNA-binding protein HF-I, known as a host factor for phage Qb RNA replication, is essential for rpoS transla- tion in Escherichia coli. Genes Dev 10, 1143–1151. 3 Roop RM II, Robertson GT, Ferguson GP, Milford LE, Winkler ME & Walker GC (2002) Seeking a niche: putative contributions of the hfq and bacA gene pro- ducts to the successful adaptation of the brucellae to their intracellular home. 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J Biol Chem 250, 3607–3612. 50 Raynal LC, Krisch HM & Carpousis AJ (1996) Bacter- ial poly(A) polymerase: an enzyme that modulates RNA stability. Biochimie 78, 390–398. M. Folichon et al. Hfq binding to RNA stimulates elongation by PAP I FEBS Journal 272 (2005) 454–463 ª 2004 FEBS 463 . Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails Marc Folichon, Fre ´ de ´ ric. 5¢ end is not critical for Hfq binding The idea that stimulation of poly(A) synthesis is cor- related with the association of Hfq to the 3¢ end of RNA