Bacterial poly(A) polymerase: An enzyme that modulates RNA stability

Biodtimie (1996) 78, 390-398

© Soci~t6franqaisede biochimieet biologic moleculaire/ Elsevier, Paris

Bacterial poly(A) polymerase: An enzyme that modulates RNA stability LC Raynal, H M Krisch, AJ C a r p o u s i s * Laboratoire de Microbiologie et GEn~tique MolEculaire, UPR 9007. CNRS. ! 18, route de Narbonne. 31062 Touhmse codex France

(Received 26 April 1996; accepted 12 June 1996)

Summary - - We have constructed a strain that overexpressesE colt poly(A) polymerase (PAP I). The recombinant protein was soluble, and a partially purified extract had high levels of poly(A) polymerising activity.An antiserum raised against the overexpressed PAP ! has permitted two types of analysis: the identification of other E colt proteins that may interact with PAP I, and the search tot PAP l-like pr~teins in other bacteria. Immunoprecipitationexperiments suggest that PAP I is associated with a 48-kDa protein. This protein remains to be identified. Western blotting using the antiserum against E colt PAP 1 revealed related proteins in a variety of Gr~:m-negativebacteria aa~din B subti/is. A comparison of the E colt protein with putative poly(A) polymerases recently identified in H influ, n:a and B subtilis showedhighly conserved ,~equencesin the amino terminal and central portions of the proteins tb~t may be important for enzyme activity. poly(A) polymerase / RNA polyadenylation / RNA degradation / RNA degradosome Introduction The amino terminal sequencing of E colt poly(A) polymeruse showed that it is encoded by the pcnB gone [ 11. PcnB (l/lasmid copy number) has a role in the regulation of the replication of CoiEI plasmids [2--4]. Mutations in pcnB reduce plasmid copy number and strains with a disrupted pcnB gone are viable but they do not stably maintain ColEI plasmids [4, 5]. PcnB mutations increase the stability of RNAI, an antisense regulator of ColEI plasmid replication [6, 71. RNAI hybridises to and inactivates RNAII, which is the primer that initiates plasmid replication (for reviews see [8, 91). Polyadenylation modifies the stability of RNAI and has been shown to have a similar role for certain E colt mRNAs [10--121. Recently a second poly(A) polymerase activity, PAP I1, was identified in an E colt strain with a deletion of the pcnB gone [13]. Little is known about the role of PAP II in RNA metabolism. The enzyme studied here, the product of the pcnB gone, is now named PAP 1. Poly(A) polymerase activity was first identified in E colt 35 years ago [14]. Although several credible reports of mRNA 3' polyadenylation in bacteria have been published (see references in [11), the ubiquitous presence of poly(A) tails in plant and animal cell mRNAs has lead to the belief that polyadenylation is important mainly in eukaryotes. Messenger RNA 3' end formation in the eukaryotic nucleus involves an endonucleolytic cleavage of the nascent transcript mediated by a protein complex (CPSE cleavage and 12olyadenylation specificity _factor) which then activates

*Correspondence and reprints

polyadenylation (for reviews see 115-17l). Eukaryotic poly(A) polymerase, which in contrast to the bacterial enzyme has little activity by itself, requires CPSF for activity. In the eukaryotic cytoplasm, the poly(A) tail is important for translation and mRNA stability, Message RNA decay is generally believed to be proceeded by the shortening of poly(A) tails. However, the details of the mechanism of eukaryotic mRNA degradation remain obscure. Bacterial mRNA decay depends on the action of both endo- and exoribonucleases 118, 191. Two exonucleases, RNase 11 and PNPase, have important roles in decay. Both these enzymes degrade RNA progressively in the 3' to 5' direction. However, 3' RNA stem-loop structures can protect RNA from attack by exonucleases I20, 211. Polyadenylation may destabilise mRNA with 3' stem-loops by adding a single-stranded tail that facilitates attack by these exonucleases. In vitro e,~:periments have shown thai polyadenylation can stimulate the degradation of the RNAI by PNPase [10]. Recent work has revealed that PNPase is part of the E colt RNA degradosome, a multienzyme complex that also contains RNase E and RhlB I22-24], RNase E is an endoribonuclease that has an important role in RNA processing and degradation while RhIB is a 'DEAD' box RNA helicase [25]. These helicases, which are highly conserved in both prokaryotes and eukaryotes, use the energy of ATP hydrolysis to unwind double-stranded RNA. Because of this hellcase activity, the bacterial RNA degradosome can efficiently degrade highly structured RNAs. The stability of bacterial messages ranges from less than 2 to greater than 20 rain. In addition to attack at the 3' end by the exonucleases RNase II and PNPase, endonucleases such as RNase E can initiate degradation by cleaving a message internally [26-29]. There is also evidence for

391 decay that is initiated by RNase E cleavage at the 5' end of certain RNAs [30], The RNA degradosome has a central role in these events. Because polyadenylation facilitates digestion by PNPase, we investigated the possibility that PAP I and the degradosome might interact. Preparations of partially purified RNA degradosome have 3' polyadenylating activity (unpublished results). However, this activity can be separated from the RNA degradosome by sedimentation in glycerol gradients containing 0.5 M NaC1. Thus poly(A) polymerase is not an integral component of the RNA degradosome. Nevertheless, it is conceivable that the degradosome interacts v ith PAP I to target poly(A) addition. Such a role is consistent with results which show that RNAI degradation depends on the RNase E component of the degradosome cleaving at the 5' end of the molecule [30, 31 ]. In this report, we describe the overexpression of E coli PAP I using a pET-based expression system. Extracts were prepared in which PAP I was 5-10% of the total soluble protein. The activity of the recombinant PAP I was similar to that previously reported for the enzyme. RNA binding was analysed using Northwestern blots. Antibodies were raised against the recombinant PAP I, and an immunoprecipitation using radiolabelled extracts showed that PAP I co-precipitates with a 48-kDa protein. This result raises the interesting possibility that PAP I might form a complex with another protein involved in_ ",he regulation of RNA stability. The antiserum against E coli PAP I detected PAP I-like proteins in bacteria as distant as B subtilis in Western blots. Comparisons of the sequences of B subtilis, H influenza and E eoli PAP I proteins revealed that extensive regions of the amino terminal and central portions of the proteins are conserved. The residues in these conserved regions are likely to be important for the function of bacterial poly(A) polymerase.

Materials and methods Plasrnid construction The E eoli pcnB open reading frame was amplified by PCR with Pfu DNA polymerase (Stratagene) using two oligonucleotide primers, 5' ATATGACACTACCGAGGTGTACTATTTTTACC 3' and 5' CTACGACGTGGTGCGCG'ITrGC 3', that hybridised to each end of this reading frame. The PCR reactions were performed following the conditions recommended by Stratagene. Thirty cycles (30 s, 94°C; 2 rain, 50°C; 3 min, 72°C) were carried out with genomic DNA from E coli DI0 that had been denatured with NaOH. The blunt-end 1.4 kb PCR product was inserted into pUC 19 vector that had been digested with Sinai. The insertion into the SmaI site creates an NdeI site that included the translation initiation codon. After transformation of the DHSa strain, the recombinant vector was purified and digested with NdeI and BamHI. The 1.4-kb fragment was ligated to pETI Ia [32] digested with the same restriction enzymes. DH5o~ was transformed with this construction. The recombinant plasmid (pPAP) was purified then transformed into the strain BL21 (DE3).

Poly¢A) polymerase expression and preparation ~[ extracts LB medium containing 50 lag/mL ampicillin was inoculated with a single fresh colony and incubated with aeration at 37°C. Expression was induced at an OD6oo= 0.4 by the addition of IPTG ( i mM final concentration). After 2 h, the cells were collected, concentrated 6-20-fold in a buffer containing 50 mM Tris-HCI (pH 8.0), 1 mM EDTA, 200 mM NaCI, 0.1 mg/mL lysozyme, I mM PMSE 10% glycerol, 2 lag/mL aprotinin, 0.8 p.g/mL leupeptin and 0.8 ~g/mL pepstatin. Crude lysates, prepared by a freeze-thaw step followed by sonication, were adjusted to 0.2% Triton X-100 and 10 mM magnesium acetate, then incubated on ice for 30 min with DNase I (10 [.tg/mL). The extracts were adjusted to 0.8 M NH4CI and incubated for another 15 min on ice. A 10 000 g supematant was prepared by centrifugation at 14 000 rpm for l 0 rain in a Sigma 20ira centrifuge. A 100 000 g supematant was prepared by centrifugation at 70 000 rpm for I h, with a TN-100 rotor in a Beckman TL- 100 ultracentrifuge. Centrifugation was at 4°C. For protein labelling, colonies were grown in a MOPS-gLucose medium [33], containing 5 p.g/mL methionine, to OD600 = 0.2 then IPTG was added. After 15 min, 35S-methionine (Amersham, SJ 1015) was added (40 p.Ci/mL). Incubation was continued for another 2 h. Extracts were prepared as described above except that the sonication step was omitted.

Protein gels and Western blot SDS-PAGE and Western blotting were performed essentially as described [34]. Proteins were separated by 9% SDS-PAGE. The gels were stained with Coomassie blue or electrotransfered to nitrocellulose (Amersham, High Bond C Extra) for Western blots. 40 p.L of a six-fold concentrated extract were loaded per lane. After migration, proteins were electroblotted in a Tris-glycine transfer buffer for 2 h at 80 mA then 2 h at 240 mA. After 30 min of blocking with 5% skim milk in 1 × PBS, the membranes were incubated 1 h with PAP I antiserum or pre-immune serum diluted in a buffer containing 0.5% skim ~ailk, 0.025% Tween 20, i × PBS, washed with the same buffer, incubated l h with the secondary amibody which was anti-rabbit IgG peroxidase conjugale, then washed. Detection was with an ECL kit (Amersham) and Biomax film (Kodak). To detect PAP I in E coil we used a 1/10 000 diluted serum, and the exposure varied between 3 rain and more than I h. The detection of PAP I in other bacterial strains was with serum diluted I]5000.

In vitro transcription RNAs were synthesised by run-off transcription using the plasmids pAC301 and pAC401 which contain portions of bacteriophage T4 gene 32 transcription unit ([35], see [36] for map of the gene 32 transcription unit), pAC301 digested with Mspl yields a 222 nt RNA, AC301M RNA. pAC401 digested with HindllI restriction enzyme, yields a 882 nt RNA, AC401H RNA. in vitro transcription was with T7 RNA polymerase (Promega; 80 U for 1 h then 40 U for another h), in a 45 laL reaction volume with a-32p-CTP or o~35S-ATP (Amersham, 50 laCi per reaction) and 1 lag of DNA of template using the conditions specified by Promega. Template DNA was digested by treatment with 1 U of DNase I (Promega). The reaction was then extracted with pbenol/chloroform/isoamyl alcohol (25/24/1). The RNAs, which were de-salted with a Sephadex G25 column equilibrated with water, were stored at -20°C.

392

Poly(a) polymerase assays

Results and discussion

RNA elongation

Overproduction orE coli poly(A) polymerase using a pET vector

The AC301M RNA labelled with ~t-3-~P-CTP(25 000--30 000 dpm) and 2 lag of competitor yeast RNA were incubated with the 100 000 g supematant of a pPAP extract (50 ng PAP I) in a 100 laL reaction containing 10raM Tris-HCl (pH 7.5), 100 mM NaCI, 1% glycerol, 0.1% Triton X-100, 4 mM MgC12, 0.2 mM EDTA, 0.2 mM DTT, and 200 laM ATP, at 37°C. At various times, 10 laL samples were withdrawn, quenched and treated with proteinase K as described previously [22]. The RNAs were separated by electrophoresis on denaturing gels (6%, 29:1; 7 M urea; 0.5 x TBE). The gel was dried and autoradiographed on Biomax film (Kodak).

Poly(A)synthesis The conditions were as described above but yeast RNA was the substrate and the radioactive AC301M RNA was omitted. 1 laCi of ~-3sS-ATP was added per 10 laL. A series of dilutions of the I00 000 g supematant of the pPAP and p~zTlla extracts were assayed in the range where less than 20% of the radioactive ATP was incorporated into TCA precipitable material. The incorporation of I nmol of,,xMP in 10 min at 37°C was taken as one unit of activity. The reaction, which was stopped by the addition of 10 pL of 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.2% SDS, and I mg/mL yeast RNA, was incubated briefly on ice before adding ! mL of cold 5% TCA. After 30 rain on ice, the precipitates were filtered on GF/C disks (Whatman).The disks were washed twice with ! mL of 5% TCA, once with 100% ethanol, dried and the radioactivity was determined by scintillation counting.

Northwestern hi,ors The procedure was as described [371. The equivalent of 100 laL of culture for pPAP (10 laL for recombinant RhlB prepared as described 1241) wexe separated by 9% SDS-PAGE and electroblotted for 2 h at 80 mA then for another 2 h at 240 mA using a sodium carbonate transfer buffer [38]. Renaturation was for at least 4 days at 4°C. The membranes were blocked for 15 min then incubated at 44~C |br 90 min with ~-3~S.ATPlabelled RNA substrate (5 x 105 cpm/mL), The blots were then washed, dried and autoradiographed with a Fuji imaging plate.

Immunoprecipitation The reaction was performed with the 100 000 g supematant of a 3-~S-methioninelabelled protein extract. 40 I.tLof a 20-fold concentrated extract was incubated with 4 vL of PAP I antisem~nor preimmune serum and 360 laL of 10 mM Tris-HCI (pH 7.5), 300 mM NaCI, ! mM EDTA, 5% glycerol, 0.2% Triton, 0.2 mg/mL BSA, 0.1 mM DTT, 0.1 mM PMSE 2 lag/mL aprotinin, 0.8 lag/mL leupeptin, 0.8 gg/mL pepstatir!, for I h at 4°C. Protein A beads (Pharmacia), 100 laL (20% bed volume in immunoprecipitation buffer), were added and incubation was continued for I h at 4°C with gentle agitation. The beads were washed with the same buffer then incubated at 95°C for 2 rain in 100 laL of SDS-PAGEgel sample buffer to release the radioactive proteins. The immunoprecipitatewas then separated by 9% SDS-PAGE. The gel was dric then autoradiographed with a Fuji imaging plate or a Biomax film (Kodak).

We have constructed a derivative of pET I 1 that overproduces E coli poly(A) polymerase (PAP I). A previous study on the purification, sequencing and overexpression of PAP I showed that the amino terminal residue is a lysine [I]. Inspection of the DNA sequence of the pcnB gene showed that there was no nearby AUG upstream of the lysine codon in any reading frame. It seemed likely that translation started at a UUG 17 codons upstream of this lysine, and that the precursor protein was cleaved at the lysine residue since a recombinant protein initiated at the position of the UUG codon was at least as active as the processed form. A shorter protein initiated at an first in-frame AUG residue downstream of the lysine codon was not active. In our construction, the entire pcnB coding sequence, beginning at the UUG codon, was amplified by PCR from genomic E coli DNA and ligated to pETI la, a plasmid vector which contains the bacteriophage T7 genelO promoter and translation initiation region [32]. In the recombinant plasmid (pPAP), the UUG initiation codon was converted to an AUG. Figure la shows a Coomassie blue stained gel of a total lysate containing the overexpressed protein. In the pPAP lane, a protein of the expected size for PAP I, 53 kDa, was strongly overexpressed. The pETI la lane shows a control cell extract prepared from BL21 (DE3) containing the parent plasmid. The level of PAP I expression using the pET system is comparable to that previously obtained using the plasmid pREI-1, which contains bacteriophage lambda PL expression signals [I ]. We constructed the pET-based system because, in our hands, it was dil'ficult to maintain the strain containing pREI-! (unpublished results). Poly(A) polymerase activity was assayed as described in Materials and methods using a 100 000 g supematant. PAP I constituted about 10% of the total protein in these extracts. Two types of assay were used. In one, we measured the synthesis of the radioactive poly(A) tails by TCA precipitation. We estimate the PAP I activity in the 100 000 g supernatant to be 250 U/mg. This activity, which is based on a simple assay using yeast RNA as substrate, is several-fold less than the 1100 U/rag obtained with a 100 000 g supernatant prepared from the strain containing the plasmid pREI-I [1]. This higher activity is probably due to differences in assay conditions. In a second assay, we used denaturing polyacrylamide gel electrophoresis to measure the increase in size of a radiolabelled RNA substrate. Figure 2a shows a time course of the polyadenylation of a 222 nt RNA. The control extract was prepared from the BL21 (DE3) containing the pETI la vector (fig 2b). After 2 rain of incubation with the pPAP extract (lane 4), most of the radioactive RNA was converted to a large product ranging from 400 to 800 nucleotides in length. The in vitro synthesised poly(A) tails are clearly much larger than the 20 to

393 50 nt added in vivo [12]. We observed some polyadenytation even in the absence of exogenous ATP (lane C). However, since the supernatants were not dialysed, this activity is probably due to traces of ATP in the extracts. No p~ecautions were taken to eliminate the RNase activity in these extracts and it is evident, especially in the pET1 la control, that they slowly degrade the radioactive RNA substrate. In a trial purification of the recombinant PAP I, we obtained enzyme that was about 90% pure by weight (data not shown). An estimation of the native size of the enzyme by gel permeation chromatography indicated that the PAP I was heterogeneous, ranging from monomers to very large aggregates. All forms of the enzyme were active. We could not disrupt the aggregates by increasing the salt concentra-

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Fig 2. Time course of RNA elongation. Exlracts ~100 900 g) prepared ariel"induction of E cHi BL21 (DE3) containin', pPAP (a) or pET I l a (b) were incubated with "~'P-iabelledRNA (.~C301M, 222 nt), competitor yeast RNA and ATP. Samples were withdrawn and the reaction stopped at 0.25, 0.50, i.0, 2.0, 3.0, 4.0, 8.0, 12 and 16 rain (lanes I--9). The products were separated by denaturing gel electrophoresis. Autoradiography was for 15 I1 at room temperature. Lane C corresponds to a 16-rain reaction in the absence of exogenously added ATE The position and size of the RNA substrate and the elongated product are indicated by horizontal arrowheads.

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Fig 1. Poly(A) polymerase expression and Western blot analysis. a. Coomassie stained 9% SDS-PAGE of crude lysates prepared after induction of protein synthesis. M, molecular mass makers; pPAP, E colt BL21 (DE3) with the plasmid pPAP which contains the E colt poly(A) polymerase gene under the control of an inducible T7 promoter; pETI la, E colt BL21 (DE3) with the plasmid pETI la, the parent vector that does not contain an insert, b, e. Western blot analysis using crude lysates from the BL21 (DE3) containing pPAP or pET1 la. The membranes were probed with antiserum against PAP I (I) or pre-immune serum (pl). The lanes in each panel come from different membranes which were exposed for the same time. The exposures are very different between b (I x, 3 min) and e (> 20 x, more than 60 min). The asterisk corresponds to the dimer of PAP I. The small arrow corresponds to the position of the endogenous PAP I which is slightly smaller than the recombinant protein (see text).

tion or by adding a non-ionic detergent. PAP I has previously been reported to have a tendency to aggregate [1 ]. For the remainder of the work described here, we used either crude lysates, or 100 000 g supernatants prepared as described in Materials and methods.

Northwestern blot analysis of RNA binding E colt poly(A) polymerase has been prepared commercially for many years following a published protocol [39]. The commercial enzyme, whi.ch has been shown to be the product of the pcnB gene [5], is known to act on a broad range of RNA substrates in vitro. In order to investigate the possibility that PAP I might have specificity for certain sequences

394 or structures, RNA binding was analysed by Northwestern blot analysis. Total extracts were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and the protein renatured on the filter. The result obtained with two different RNA substrates is shown in figure 3. The AC401H and AC301M RNAs are from different regions of the bactet'~ophage T4 gene 32 transcription unit [35, 36]. The b,C40 i H RNA contains the downstream two-thirds of the gene 32 tra~iscript including the stem-loop of the rho-independent transcription terminator. The AC301M RNA contains the dista~ portion of orfC which is just upstream of the gene 32 RNase E processing site. Both of these RNAs bind to the overexpressed PAP I (fig 3a, b, lane 1). Several other

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RNA binding proteins detected by this methot, were also present in the pETI la control extract (fig 3a, b, lane 2). Each panel shows a control reaction (lane R) using recombinant RhlB which is known to bind RNA using this protocol [24]. Several other RNAs from the gene 32 transcription unit were tested (data not shown), including substrates with the '-71' RNase E processing site and mutants that alter an RNA stem-loop structure at this site [35]. All of the RNAs bind to PAP I and no substrate specificity was detected. The PAP I also bound radioactive double-stranded DNA but ill all cases binding was effectively competed by the addition of yeast RNA (data not shown). These results, which suggest that PAP I has a general affinity for nucleic acids, provide no evidence that the enzyme has a preference for binding to a particular RNA sequence or structure.

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Fig 3. Northwestern blot analysis. Blots with renatured proteins were probed with various ~]2 P-tahelled RNAs. Autoradiographv was for 2 h with a Fuji imaging plate, a. AC401H RNA. b. AC30 ! M RNA. In each panel: lane I, crude lysates prepared after induction of BL21 (DE3) containing pPAP; lane 2, pETI la control; and lane R, extract containing overexpressed RhlB. The arrow marks the position of PAP ! and the asterisk (*) the position of a dimer of PAP I. The position of RhlB (50 kDa) is marked with the solid square (B). The position and size of three unidentified proteins which bind RNA are indicated by horizontal arrowheads.

We originally raised rabbit antibodies against PAP ! overproduced using the pRE l-1 plasmid obtained from Sarkar [ 1]. Although Western blots employing this antiserum could detect overexpressed PAP I, they did not detect the low level of endogenous PAP I normally present in the cell. In order to obtain a better antiserum, several hundred micrograms of PAP I, produced from pPAP, were purified by SDS-PAGE and used to immunise a rabbit. Western blots using this antiserum are shown in figure 1 (b, c). The overexpressed PAP I was easily detected in figure lb. The 100-kDt protein marked with an asterisk in figure lb is apparently a dimer of PAP I. This form is present even though the sample was boiled for 5 rain in a loading buffer containing 2% SDS and 5% mercaptoethanol. In figur,~ lc, with a much longer exposure, it is possible to detect the endogenous PAP I which is slightly smaller than the overexpressed protein. Thus, as expecled, the endogenous PAP I corresponds to the processed protein which is 17 amino acids shorter.

lmmunoprecipitation of poly(A ) polymerase Recently, the role of the interactions between the enzymes involved in the processing and degradation of bacterial RNA has begun to be appreciated [22-24]. There appears to be a functional interaction between the RNA degradosome and PAP I [10, 40]. This connection prompted us to investigate if PAP I exists as an independent enzyme or as part of a protein complex. Figure 4 shows the results of an immunoprecipitation using PAP I antibody, and 35S-methionine labelled extracts prepared from the pETI la strain. The endogenous PAP I was detected as well as another protein that migrates slightly faster. It seems unlikely that the clearly resolved doublet in figure 4 is due to the presence of a faster migrating, degraded or modified form of PAP I. These results raise the interesting possibility that PAP I exists in the cell as part of complex with another protein.

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Fig 4. Immunoprecipitation with antibody against poly(A) polymerase. The reaction] using pre-immune serum (pI) or PAP I antiserum (I), was with a ~S-methionine labelled extract (I 00 000 g) prepared afi.er induction of E co/i BL21 (DE3) containing pETI la. The immunoprecipitates were separated on 9% gels. The gel was autoradiographed with a Fuji imaging plate for 4 days. The positions of the endogenous PAP I ond another protein that co-precipitates (X) are indicated to the right of the figure.

protein in B subtilis is noteworthy because this Gram-posi: ve bacterium separated from E coil more than one billion years ago [41]. The blots in figure 4 were heavily overexposed to permit visualisation of the reaction of the E coil antibody with the hemologue in other bacteria. A series of shorter exposures showed that the signals from the other bacteria were about 10-fold lower than from E coil (data not shown). Messenger RNA 3' polyadenylation h~s been observed in many bacteria, including B subtilis (see references in [42]). The detection of a PAP I-like protein in B subtilis suggests that this enzyme is conserved in a wide range of bacterial species. The analysis of the H it~uenza genome, which has now been completely sequenced, shows that it has genes encoding proteins that are very similar to E coil PAP I, RNase II, RNase III, RNase D, RNase E, RNase P, RNase PH, PNPase, RhlB, and tRNA nucleotidyltransferase [43]. RNase E-like proteins have been detected, by Western blotting, in a variety of Gram-negative bacteria [44], and there is evidence for RNase E activity in the photosynthetic bacteriurr R capsulatus [45]. PNPase activity is found in both prokaryotes and eukaryotes, and a protein with PNPase activity, which is 43% identical to E coli PNPase, has recently been identified in the spinach chloroplast [46]. These surprising results suggest that many of enzymcs involved in RNA processing and degradation may be conserved throughout the bacterial kingdom.

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We are currently attempting to identify the 48-kDa protein associated with PAP I. Detection of PAP ~-like proteins in other bacte.,'ia. Our antiserum against PAP I was used to search for related proteins in other bacteria. The identification and characterisation of related enzymes could be useful in defining those motifs that are essential for enzyme activity. Figure 5 shows a Western analysis in which pre-immune and antiPAP I serum were used to probe blots of proteins from five Gram-negative bacteria Y pseudotubercuiosis, E carotovora, S marcescens, P mirabilis and D gigas, and a Grampositive bacterium, B subtilis. A comparison of panels a (pre-immune) and b (anti-PAP I) shows that, except for S marcescens, a protein between 45 and 55 kDa reacts specifically with the E coil PAP I antiserum. There is not a clear signal in lane 8 for S marcescens, which is surprising considering that this species is more closely related to E coil than P mirabilis, D gigas or B subtilis. The detection of the

Fig 5. Western bin, analysis of proteins t'rom a variety of bacteria. Crude protein extracts were separated by SDS-PAGE, blotted and reacted with pre-immune (pl) (a) or anti-E co/i-PAP I serum (1) (b). Lanes ! to 8: endogenous PAP I (E toll), overexpressed PAP 1 (pPAP in E coil), Bacillus subtilis, Desulfovib;'io gigas, Yersinia pseudomberculosis, Erwinia carotovora, Proteus mirabi/is, and Serratia malz'es('ens, respectively. Positions of the different homoIogues detected are indicated on the figure by vertical arrows.

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Protein sequence comparisons

Analysis of the H influenza genome revealed an open reading frame encoding a protein that is 49.5% identical to E coli PAP I [43]. We learned from A Sorokin that there is a putative B subtilis protein that is 18.9% identical to E coli PAP I [47]. Figure 6 shows an alignment of the E coli, H

431 446 358

,

GEFQVSAPPDQKGIVN~LDE-EPSPRQSYSSSTQTRT ..... ~S--HEYQFSNGEQREQLIQ~QRLHPKPKKKYYRPRRRKT ..... ~CSAE --LALRNRPAGKWVSE~LQWIEQAWTGKLSNQKKHIEEWLK~CGQH . ,

381 318

469 488 403

Fig 6. Comparison of E coli poly(A) polymerase with homologous proteins from H it!fl.en'.a and B subtilis, Comparisons were made using the program CLUSTAL I49, 501 in PC/Gene. The PAP 1 sequences were from E coil [ I I, H influe.za [431 and B subtilis I471. Conserved residues are marked with the light and dark grey boxes. The dark grey boxes indicatethe subset of residues that are conserved between the three poly(A) polymerases and tRNA nucleotidyltransferase from E coli and H il~uenza. The dots mark positions were the residues are similar between the PAP I homologues. Similarity was defined according to the matrix of Dayhoff ([49, 50] and references cited therein).

influenza and B subtilis protein sequences. The amino terminal and central portions of these proteins are highly conserved. It was previously shown that there are similarities between E coli PAP I and nucleotidyltransferase, an enzyme that adds the 3' terminal CCA to tRNA I48]. An alignment of the PAP I sequences the E coil and H influenza tRNA nucleotidyltransferase confirmed this relationship (data not

397 shown). The residues marked in dark grey in the sequence comparison of figure 6 are perfectly conserved between the three poly(A) poJymerases and the two nucleofidyltransferase proteins. These results suggest that the two enzymes share a common structure and/or mechanism of catalysis. These conserved residues are potential targets for a mutational analysis of the pcnB gene. The characterisation of the effect of these mutation on PAP I activity could facilitate the definition of the critical domains involved in the function of the enzyme.

Acknowledgments We thank N Sarkar for the plasmid pREI-1, P Dennis for suggesting MOPS-glucose medium for labelling cells with 3-~S-methionine and for encouraging us to do the 'zoo' blot, A Sorokin for informing us that a gene encoding a PAP I homoiogue was recently identified in B subtilis, Y de Preval, D Villa, A Franc and F Rodriguez (IBCG, Toulouse) for oligonucleotide synthesis, photography, and computer services. LCR is the recipient of a pre-doctoral fellowship from the MESR. This research was supported by the CNRS. Additional support was by grants from the ARC and the EU Human Capital and Mobility Program.

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