The CafA Protein Required for the 5′-Maturation of 16 S rRNA Is a 5′-End-dependent Ribonuclease That Has Context-dependent Broad Sequence Specificity
2000; Elsevier BV; Volume: 275; Issue: 12 Linguagem: Inglês
10.1074/jbc.275.12.8726
ISSN1083-351X
AutoresMark R. Tock, A. P. Walsh, Gregory T. Carroll, Kenneth J. McDowall,
Tópico(s)RNA Research and Splicing
ResumoThe CafA protein, which was initially described as having a role in either Escherichia colicell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5′-end of 16 S rRNA. The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E. coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids. We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting. We detected CafA cleavage of RNAI and a structured region from the 5′-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA. Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs. Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro. We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5′-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5′-end of an RNA. Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that CafA can cut RNAI andompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E. coli. The CafA protein, which was initially described as having a role in either Escherichia colicell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5′-end of 16 S rRNA. The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E. coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids. We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting. We detected CafA cleavage of RNAI and a structured region from the 5′-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA. Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs. Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro. We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5′-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5′-end of an RNA. Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that CafA can cut RNAI andompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E. coli. untranslated region nucleotide(s) The overproduction of CafA (1.Wachi M. Doi M. Ueda T. Ueki M. Tsuritani K. Nagai K. Mastuhashi M. Gene (Amst.). 1991; 106: 135-136Crossref PubMed Scopus (23) Google Scholar, 2.Wachi M. Doi M. Tamaki S. Park W. Nakajima-Iijima S. Matsuhashi M. J. Bacteriol. 1988; 169: 4935-4940Crossref Google Scholar, 3.Wachi M. Doi M. Okada Y. Matsuhashi M. J. Bacteriol. 1989; 171: 6511-6516Crossref PubMed Scopus (97) Google Scholar) under conditions of slow growth has been shown to cause the formation of chained cells and minicells. The presence of the latter has been interpreted as evidence for CafA either enhancing the rate of cell division and/or inhibiting chromosome partitioning after replication (4.Okada Y. Wachi M. Hirata A. Suzuki K. Nagai K. Matsuhashi M. J. Bacteriol. 1994; 176: 917-922Crossref PubMed Google Scholar). Electron microscopic examination of the chained cells revealed axial filamentous bundles, termedcytoplasmic axial filaments (hence the designation CafA), running through the center of their cytoplasms. Furthermore, the cytoplasmic axial filaments appear to be composed almost entirely of CafA (5.Okada Y. Hirata A. Matsuhashi M. Shibata T. J. Cell. Biochem., Suppl. 1995; 19 (abstr.): 114Google Scholar). This finding combined with the phenotype of cells overproducing CafA has led to the proposal that in normal cells these filaments in an unbundled form may have a role as cytoskeletal-like elements in either cell division or chromosome segregation (4.Okada Y. Wachi M. Hirata A. Suzuki K. Nagai K. Matsuhashi M. J. Bacteriol. 1994; 176: 917-922Crossref PubMed Google Scholar). The sequence of CafA has 34% similarity with the N-terminal nucleolytic domain of RNase E (6.McDowall K.J. Hernandez R.G. Lin-Chao S. Cohen S.N. J. Bacteriol. 1993; 175: 4245-4249Crossref PubMed Scopus (102) Google Scholar), an essential Escherichia coli ribonuclease that is required for the generation of 5 S rRNA from a 9 S precursor (7.Gegenheimer P. Watson N. Apirion D. J. Biol. Chem. 1977; 252: 3064-3073Abstract Full Text PDF PubMed Google Scholar) and has a central role in the decay and/or processing of a variety of RNAs, including many if not most mRNAs and RNAI, the antisense RNA regulator of the replication of ColE1-type plasmids (for reviews see Refs. 8.Cohen S.N. McDowall K.J. Mol. Microbiol. 1997; 23: 1099-1106Crossref PubMed Scopus (123) Google Scholar and 9.Coburn G.A. Mackie G.A. Prog. Nucleic Acid Res. Mol. Biol. 1999; 62: 55-108Crossref PubMed Scopus (267) Google Scholar). Endoribonucleolytic cleavage by RNase E occurs within single-stranded A and/or U-rich segments (10.Mackie G.A. J. Biol. Chem. 1992; 267: 1054-1061Abstract Full Text PDF PubMed Google Scholar, 11.Ehretsmann C.P. Carpousis A.J. Krisch H.M. Genes Dev. 1992; 6: 149-159Crossref PubMed Scopus (184) Google Scholar); however, there is no simple relationship between the order of nucleotides and the phosphodiester bond(s) that is cleaved (12.McDowall K.J. Lin-Chao S. Cohen S.N. J. Biol. Chem. 1994; 269: 10790-10796Abstract Full Text PDF PubMed Google Scholar, 13.Lin-Chao S. Wong T.T. McDowall K.J. Cohen S.N. J. Biol. Chem. 1994; 269: 10797-10803Abstract Full Text PDF PubMed Google Scholar). An oligonucleotide corresponding in sequence to the 5′-end of RNAI has been found to be cut efficiently by RNase E in vitro (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar). Combined with the knowledge that alteration of secondary structures adjacent to RNase E sites can either increase or decrease the rate of cleavage (11.Ehretsmann C.P. Carpousis A.J. Krisch H.M. Genes Dev. 1992; 6: 149-159Crossref PubMed Scopus (184) Google Scholar, 14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar, 15.Cormack R.S. Mackie G.A. J. Mol. Biol. 1992; 228: 1078-1090Crossref PubMed Scopus (75) Google Scholar), this finding has contributed to the notion that secondary structures within complex RNAs rather than serving as direct recognition motifs (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar, 15.Cormack R.S. Mackie G.A. J. Mol. Biol. 1992; 228: 1078-1090Crossref PubMed Scopus (75) Google Scholar) affect RNase E cleavage by either limiting access of the enzyme (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar, 16.Bouvet P. Belasco J.G. Nature. 1992; 360: 488-491Crossref PubMed Scopus (187) Google Scholar, 17.Mackie G.A. Genereaux J.L. Masterman S.K. J. Biol. Chem. 1997; 272: 609-616Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and/or determining the stability of local structures, which in turn determines the single-strandedness (and thus cleavability) of susceptible sites (15.Cormack R.S. Mackie G.A. J. Mol. Biol. 1992; 228: 1078-1090Crossref PubMed Scopus (75) Google Scholar, 18.Naureckiene S. Uhlin B.E. Mol. Microbiol. 1996; 21: 55-68Crossref PubMed Scopus (31) Google Scholar, 19.Mackie G.A. Genereaux J.L. J. Mol. Biol. 1993; 234: 998-1012Crossref PubMed Scopus (59) Google Scholar). Recently, it has been shown that efficient cleavage by RNase E is dependent on the nature of the 5′-end of its substrates. Compared with a linear substrate that had a 5′-monophosphate group immediately followed by a single-stranded segment, circular substrates, 5′-triphosphorylated substrates, or 5′-monophosphorylated substrates that had a duplex at the extreme 5′-end were found to be cut inefficiently by RNase E in vitro (20.Mackie G.A. Nature. 1998; 395: 720-723Crossref PubMed Scopus (342) Google Scholar). The two latter observations are consistent with the in vivo findings that pppRNAI−5 is decayed more slowly than pRNAI−5(21.Lin-Chao S. Cohen S.N. Cell. 1991; 65: 1233-1242Abstract Full Text PDF PubMed Scopus (178) Google Scholar) and 5′-stem-loops (or rather the absence of an unpaired segment) can stabilize RNA (16.Bouvet P. Belasco J.G. Nature. 1992; 360: 488-491Crossref PubMed Scopus (187) Google Scholar, 22.Emory S.A. Bouvet P. Belasco J.G. Genes Dev. 1992; 6: 135-148Crossref PubMed Scopus (230) Google Scholar), respectively. Additionally, the preferential cleavage by RNase E of 5′-monophosphorylated RNAs, such as downstream fragments produced by this enzyme (e.g.pRNAI−5), over 5′-triphosphorylated intact RNAs (e.g. pppRNAI) suggests that once decay of an RNA molecule is initiated its completion will be preferred over the cutting of intact RNA (20.Mackie G.A. Nature. 1998; 395: 720-723Crossref PubMed Scopus (342) Google Scholar). This notion may explain the observation that in general RNAs decay without the accumulation of significant levels of decay intermediates, the so called "all or nothing" phenomenon (20.Mackie G.A. Nature. 1998; 395: 720-723Crossref PubMed Scopus (342) Google Scholar). RNase E has recently been shown to be also capable of removing 3′-poly(A) tails (23.Huang H.J. Liao J. Cohen S.N. Nature. 1998; 391: 99-102Crossref PubMed Scopus (74) Google Scholar), which are known to facilitate 3′-exonucleolytic attack by polynucleotide phosphorylase (PNPase; for reviews see Refs.24.Cohen S.N. Cell. 1995; 80: 829-832Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 25.Manley J.L. Proc. Natl. Acad. Sci., U. S. A. 1995; 92: 1800-1801Crossref PubMed Scopus (30) Google Scholar, 26.Sarkar N. Microbiology. 1996; 142: 3125-3133Crossref PubMed Scopus (57) Google Scholar, 27.Sarkar N. Annu. Rev. Biochem. 1997; 66: 173-197Crossref PubMed Scopus (168) Google Scholar). Furthermore, it was reported that the poly(A) nuclease activity of RNase E is blocked by the presence of a 3′-monophosphate group suggesting that it is 3′-exonucleolytic. Although, the precise role of the poly(A) nuclease activity of RNase E in RNA decay remains to be determined, it seems likely that any action it has on poly(A) tails in vivo would affect processing by PNPase, which together with RNase E, the RhlB helicase, and the glycolytic enzyme enolase form the core of the RNA degradosome (28.Carpousis A.J. Van H.G. Ehretsmann C. Krisch H.M. Cell. 1994; 76: 889-900Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 29.Py B. Causton H. Mudd E.A. Higgins C.F. Mol. Microbiol. 1994; 14: 717-729Crossref PubMed Scopus (202) Google Scholar). This noncovalent assembly is almost certainly the major cellular machine for the decay and processing of RNA in E. coli (for review see Ref. 30.Carpousis A.J. Vanzo N.F. Raynal L.C. Trends Genet. 1999; 15: 24-28Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Both the endonucleolytic and poly(A) nuclease activities of RNase E are located in its N-terminal half (23.Huang H.J. Liao J. Cohen S.N. Nature. 1998; 391: 99-102Crossref PubMed Scopus (74) Google Scholar, 31.McDowall K.J. Cohen S.N. J. Mol. Biol. 1996; 255: 349-355Crossref PubMed Scopus (151) Google Scholar). The C-terminal half of RNase E contains the binding sites for the other major degradosome components (32.Kaberdin V.R. Miczak A. Jakobsen J.S. Lin-Chao S. McDowall K.J. von Gabain A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11637-11642Crossref PubMed Scopus (123) Google Scholar, 33.Vanzo N.F. Li Y.S. Py B. Blum E. Higgins C.F. Raynal L.C. Krisch H.M. Carpousis A.J. Genes Dev. 1998; 12: 2770-2781Crossref PubMed Scopus (277) Google Scholar) and an arginine-rich RNA binding site (31.McDowall K.J. Cohen S.N. J. Mol. Biol. 1996; 255: 349-355Crossref PubMed Scopus (151) Google Scholar, 34.Cormack R.S. Genereaux J.L. Mackie G.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9006-9010Crossref PubMed Scopus (77) Google Scholar, 35.Taraseviciene L. Bjork G.R. Uhlin B.E. J. Biol. Chem. 1995; 270: 26391-26398Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 36.Kaberdin V.R. Chao Y.H. Lin-Chao S. J. Biol. Chem. 1996; 271: 13103-13109Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Given the extensive sequence similarity between CafA and the N-terminal domain of RNase E (6.McDowall K.J. Hernandez R.G. Lin-Chao S. Cohen S.N. J. Bacteriol. 1993; 175: 4245-4249Crossref PubMed Scopus (102) Google Scholar), we purified CafA and investigated whether it has ribonucleolytic activity. Here we report that CafA is indeed a ribonuclease, which is consistent with the recent finding that it is required for the 5′-maturation of 16 S ribosomal RNA (37.Wachi M. Umitsuki G. Shimizu M. Takada A. Nagai K. Biochem. Biophys. Res. Commun. 1999; 259: 483-488Crossref PubMed Scopus (132) Google Scholar, 38.Li Z.W. Pandit S. Deutscher M.P. EMBO J. 1999; 18: 2878-2885Crossref PubMed Scopus (239) Google Scholar). Furthermore, the results of our investigation into the nature of the ribonucleolytic activity of CafA raise the possibility that it may also have a role in RNA decay. Oligoribonucleotides BR10, BR10p, and A40 were synthesized using an ABI 391 DNA synthesizer with a modified 1-μmol DNA assembly cycle and standard ABI reagents, and the coupling efficiencies were determined by trityl assay as described previously (39.Murray J. Synthesis and Characterization of Biologically Important RNA Fragments. Ph.D. thesis. University of Leeds, Leeds1996Google Scholar). Deprotected RNAs were purified by anion exchange chromatography using a Dionex DNAPac PA-100 column (4 × 250 mm) on a Dionex DX500 high pressure liquid chromatography unit. RNAI, 9 S RNA, and the 5′-UTR1 of ompAmRNA were synthesized using the T7-MEGAshortscript from Ambion. Typically 50 nm of DNA template was incubated at 37 °C for 90 min in a 20-μl reaction as described by the vendor. When internally labeled RNA was required, 60 μCi of [α-32P]UTP (ICN) was included in the reaction. Transcripts were visualized by either UV shadowing or autoradiography (40.Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and gel purified. 9 S RNA and a segment of the 5′-UTR ofompA mRNA were generated from HaeIII-cut pTH90 (41.Sohlberg B. Lundberg U. Hartl F.U. von Gabain A. Proc. Natl. Acad. Sci., U. S. A. 1993; 90: 277-281Crossref PubMed Scopus (49) Google Scholar) and HindIII-cut p106B-64 (a gift from J.G. Belasco, Skirball Institute), respectively, whereas the template for the synthesis of RNAI was a polymerase chain reaction product generated using primers with sequences 5′-GCATCCTAATACGACTCACTATAGGGACAGTATTTGGT and 5′-AACAAAAAACCACGCTACCACCAGC. Four to five pmols of RNA that had been either chemically synthesized or transcribed in vitroand then dephosphorylated was radiolabeled at the 5′-end by incubating with 23 pmols (160 μCi) of [γ-32P]ATP (ICN) and 10 units of T4 polynucleotide kinase (MBI Fermentas) at 37 °C for 10 min in 20 μl of forward reaction buffer provided by the vendor. The reactions were quenched with urea-loading buffer (7 m urea, 0.1% (w/v) bromphenol blue, and xylene cyanol) and the 5′-labeled RNAs were gel purified. RNA that had been transcribed in vitrowas dephosphorylated by incubating 4–5 pmols with 1 unit of bacterial alkaline phosphatase (Life Technologies, Inc.) at 60 °C for 1 h in buffer provided by the vendor. The alkaline phosphatase was removed by adding 20 μg of fungal proteinase K (Life Technologies, Inc.) and incubating at 37 °C for 30 min. To remove proteinase K, this reaction mixture was extracted with phenol/chloroform and chloroform and the RNA precipitated using ethanol as described previously (40.Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Six to seven pmols of RNA was radiolabeled at the 3′-terminus in a volume of 20 μl containing 50 mm HEPES (pH 7.5), 3.3 mm dithiothreitol, 10 mm MgCl2, 10% (v/v) Me2SO, 10 units of T4 RNA ligase (Amersham Pharmacia Biotech), and 33 pmols (100 μCi) of [5′-32P]cytidine 3′,5′-bisphosphate (ICN). After incubation at 4 °C for 2 h, the reactions were quenched with urea-loading buffer, and the labeled RNAs were gel purified. Labeled full-length RNA was separated from truncated forms and unincorporated nucleotides by running in polyacrylamide sequencing gels. 8 and 20% (w/v) polyacrylamide gels were used to purify transcripts and oligoribonucleotides, respectively. An autoradiograph of the gel was used as a template, and a slice of gel containing the radiolabeled RNA was excised. RNA was eluted from the gel slice into 400 μl of 150 mm NaCl, 50% (v/v) acidified phenol (pH 4.3) at 37 °C for 2–16 h. The eluate was extracted with phenol and chloroform, precipitated using ethanol, and resuspended in water (Sigma). ThecafA gene segment was amplified from plasmid pMEL1 (42.Wachi M. Nagai K. J. Cell. Biochem. Suppl. 1993; 17E: 307Google Scholar) using an Elongase kit as per the vendor's instructions (Life Technologies, Inc.). The primers used were 5′-GCCCGGGCATATGACGGCTGAATTGTTAGTAAACG and 5′-GCGGGATCCTTACATCATTACGACGTCAAACTGC, which introduced unique NdeI and BamHI sites (boldtype) at the 5′- and 3′-end, respectively, of the cafAcoding sequence. The resulting 1.4-kilobase polymerase chain reaction product was cut with NdeI and BamHI and cloned between the corresponding sites of pET16b (Novagen). The resulting plasmid was designated pCAFA01. Cultures of E. coli BL21(DE3) cells harboring either pCAFA01 (this work) or pNSTOP (31.McDowall K.J. Cohen S.N. J. Mol. Biol. 1996; 255: 349-355Crossref PubMed Scopus (151) Google Scholar), a plasmid encoding the N-terminal ribonucleolytic domain (residues 1–498) of RNase E, were grown in 2YT (40.Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) to an A 600 of 0.6. Expression of the plasmid-cloned gene was then induced by adding isopropyl-1-thio-β-d-galactopyranoside to 1 mm. After incubation for a further 60 min at 37 °C, the cells were harvested by centrifugation at 4000 × g for 10 min using a MSE 3000 centrifuge. The cell pellet was resuspended in 50 ml of binding buffer (7 m urea, 20 mmTris-HCl (pH 7.6), 500 mm NaCl, 5 mm imidazole, 0.1% (w/v) Triton X-100), and the cells were ruptured by passage through an French Pressure Cell (Amicon). The insoluble material including intact cells was removed by high speed centrifugation (100,000 × g, 30 min) using a Beckman SW28 rotor. The supernatant was loaded on to a 5-ml nickel-charged HiTrap Chelating column (Amersham Pharmacia Biotech), which was washed with 30 ml of binding buffer before bound proteins were eluted with an imidazole gradient (5 mm to 1 m over 20 ml). All of the above purification steps were done using buffers, columns, and equipment that have been cooled to below 4 °C. The protein content of each fraction was assayed using a modified Bradford assay (Bio-Rad) and SDS-polyacrylamide gel electrophoresis. In initial experiments (Fig. 1), CafA was purified from extracts of cells from a 1-liter culture; however, we found that purification from a cell extract of a 4-liter culture resulted in reduced levels of contaminating polypeptides (Fig. 2).Figure 2Electroeluted CafA has ribonucleolytic activity. A, SDS-polyacrylamide gel electrophoresis analysis of a batch of affinity purified CafA (lane 1) that was further purified by electroelution from a preparative gel (lane 3). Lanes 2 and 4 contain samples of eluate from the gel above and below the position of CafA, respectively, whereas lane M contains 10-kDa Protein Size Markers (Life Technologies, Inc.). B, assay of samples inA for ribonucleolytic activity. 5′-Labeled BR10 was incubated with equal amounts (50 ng) of immobilized metal affinity chromatography purified and electroeluted CafA (RNase G) under conditions used in Fig. 1. The markers in this panel (lane M) were a 1-nt ladder generated by limited PNPase decay of the substrate. The source of PNPase was a preparation of degradosome that was purified and assayed as described previously (28.Carpousis A.J. Van H.G. Ehretsmann C. Krisch H.M. Cell. 1994; 76: 889-900Abstract Full Text PDF PubMed Scopus (385) Google Scholar). The numbers on the right of this panel indicate the size in nucleotides of the substrate and cleavage products.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recombinant CafA protein was further purified from a preparative Lamelli gel (40.Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) using a Mini Whole Gel Eluter (Bio-Rad). Protein was eluted from a 10% (w/v) polyacrylamide gel (7.2 × 10.0 × 0.1 cm) as per the vendor's instructions into 50 mm Tris-HCl (pH 8.7), 25 mm boric acid. A voltage of 200 V (10 watt) was applied for 30 min. At the end, the polarity was reversed for 15 s to remove any protein that might have stuck to the membrane during elution. Residual SDS was removed from the eluate samples using Extracti-gel D as per the vendor's instructions (Pierce). Two hundred ng of either CafA or RNase E was incubated with 4–15 pmols of the appropriate substrate RNA in 20 μl of 20 mm Tris-HCl (pH 7.4), 100 mmNaCl, 10 mm MgCl2, 0.1% (v/v) Triton X-100, and 1 mm dithiothreitol containing 20 units of RNase inhibitor (Amersham Pharmacia Biotech) at 37 °C for up to 60 min. Samples were taken at regular intervals and quenched using urea-loading buffer before aliquots were analyzed using polyacrylamide sequencing-type gels (40.Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Fractions across a peak of recombinant His-tagged CafA that was purified using immobilized metal affinity chromatography (Fig. 1 A) were incubated with 5′-labeled BR10 (5′-ACAGUAUUUG), a synthetic decaribonucleotide that corresponds in sequence to the single-stranded segment at the 5′-end of RNAI (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar). Ribonucleolytic activity was detected in all fractions containing CafA (B); moreover, the level of activity was directly proportional to the amount of this polypeptide (C). To confirm that CafA was the source of the ribonucleolytic activity, we further purified CafA by electroeluting, following preparative gel electrophoresis, a batch that contained reduced amounts of contaminating polypeptides (compare Fig.1 A and Fig. 2 A) as a result of increasing the amount of cell extract added to the immobilized metal affinity chromatography column (see "Experimental Procedures"). The resulting CafA preparation (Fig. 2) was homogeneous as judged by staining of SDS-polyacrylamide gels using Coomassie Blue (A) and silver (data not shown) and was still able to cleave the decanucleotide substrate (B). Thus, from here on we will adopt the designation RNase G (37.Wachi M. Umitsuki G. Shimizu M. Takada A. Nagai K. Biochem. Biophys. Res. Commun. 1999; 259: 483-488Crossref PubMed Scopus (132) Google Scholar, 38.Li Z.W. Pandit S. Deutscher M.P. EMBO J. 1999; 18: 2878-2885Crossref PubMed Scopus (239) Google Scholar) when referring to CafA. Comparison of the migration of the upstream products of RNase G cleavage with a 1-nt ladder generated using PNPase revealed that they were 5, 6, and 7 nt (B). To determine whether the products of RNase G cleavage were generated endoribonucleolytically, we used as substrate 5′-monophosphorylated BR10 labeled at its 3′-end by the addition of [5′-32P]pCp. As shown in Fig.3, downstream products of 4, 5, and 6 nt were detected (A) consistent with endonucleolytic cleavage at the same positions identified using 5′-labeled BR10. Moreover, the relative abundance of each of the downstream products (Fig.3 A) mirrored precisely that of the corresponding upstream products (Fig. 2) indicating that RNase G is only able to cut individual BR10 decanucleotides once. Incubation of RNase G with two BR10 derivatives that had either three extra Gs at the 5′-end or a C at the 3′-end resulted in cleavage at precisely the same positions (relative to sequence) observed for BR10 (data not shown), indicating that as found for RNase E (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar), the specificity of RNase G cleavage of BR10 is determined by sequence rather than a distance measured in nucleotides from either its 3′- or 5′-end. We next investigated whether efficient RNase G cleavage is dependent on the presence of a monophosphate on the 5′-end of its substrates by incubating with RNase G an aliquot of 3′-labeled BR10 that had not been 5′-phosphorylated (Fig. 3 B). We were unable to detect cleavage of this substrate by RNase G after 60 min of incubation (B) even though 50% of 5′-monophosphorylated BR10 was cut within 2 min (A). This finding indicates that like RNase E, which was included as a control, RNase G is a 5′-end-dependent ribonuclease (20.Mackie G.A. Nature. 1998; 395: 720-723Crossref PubMed Scopus (342) Google Scholar). In contrast, we found that the 3′-phosphorylation status did not affect the rate of cleavage of 5′-labeled BR10 by RNase G or RNase E; an oligonucleotide synthesized with a 3′-phosphate was cleaved as efficiently as one that had a hydroxyl group at its 3′-end (Fig.4). Having found that RNase G can cut BR10 (Fig. 3), we decided to investigate using complex substrates whether RNase G can cut within other single-stranded segments cleavable by RNase E and/or would cut at other positions. To this end, we incubated RNase G and, as a control, RNase E with RNAI, the 5′-UTR ofompA mRNA, 9 S RNA, and a segment from 23 S rRNA (Fig.5). All of the substrates used were labeled at their 5′-ends. Our RNase E preparation cut RNAI primarily at the −5 position within the single-stranded region at its 5′-end. Less efficient RNase E cleavage at the −6 position and at internal sites within RNAI was also evident as observed previously (14.McDowall K.J. Kaberdin V.R. Wu S.W. Cohen S.N. Lin-Chao S. Nature. 1995; 374: 287-290Crossref PubMed Scopus (124) Google Scholar, 36.Kaberdin V.R. Chao Y.H. Lin-Chao S. J. Biol. Chem. 1996; 271: 13103-13109Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). RNase G was also able to cut RNAI; moreover, the major sites of cutting overlapped those of RNase E within the single-stranded segment at the 5′-end (A). Relative to RNase E, however, RNase G was able to cut more efficiently at the −6 position but appeared to be unable to cleave at internal sites. In the case of the 5′-UTR segment ofompA mRNA, we found that RNase G was able to cut within two internal segments that contain major RNase E sites designated "c" and "d" (43.Lundberg U. von Gabain A. Melefors O. EMBO J. 1990; 9: 2731-2741Crossref PubMed Scopus (79) Google Scholar, 44.Lundberg U. Melefors O. Sohlberg B. Georgellis D. von Gabain A. Mol. Microbiol. 1995; 17: 595-596Crossref PubMed Scopus (10) Google Scholar), albeit more slowly than our RNase E preparation (B). Close examination of this gel suggests that although RNase G is able to cut within the c segment of ompAmRNA, the precise bonds that were cleaved differ from those cut by RNase E. We were unable to detect RNase G cleavage at the a RNase E site of 9 S RNA (C), which is required for the generation of 5 S rRNA (45.Ghora B.K. Apirion D. Cell. 1978; 15: 1055-1066Abstract Full Text PDF PubMed Scopus (209) Googl
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