Rnase E Activity Is Conferred by a Single Polypeptide: Overexpression

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9006-9010, October 1993 Biochemustry RNase E activity is conferred by a single polypeptide: Overexpression, purification, and properties of the ams/rne/hmpl gene product (endoribonuclease/RNA processing) R. S. CORMACK, J. L. GENEREAUX, AND G. A. MACKIE* Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1 Communicated by Myron K. Brakke, June 29, 1993 (receivedfor review May 10, 1993) ABSTRACT Ribonuclease E, an enzyme that processes encoding a high molecular mass protein hypothesized to be pre-SS rRNA from Its precursor, is now believed to be the involved in nucleoid partitioning or cell wall invagination major endoribonuclease participating in mRNA turnover in during division (16). The revised DNA sequence, coterminal Escherichia coli. The product of the ams/rne/hmpl gene, with the 5' end of the two previous sequences, encodes a which is required for RNase E activity, was overexpressed, protein of 1025 amino acid residues whose predicted relative purified to near homogeneity by electroelution from an molecular mass would be 114,000. This polypeptide displays SDS/polyacrylamide gel, and renatured. The purified poly- anomalous electrophoretic mobility in SDS/polyacrylamide peptide possesses nucleolytic activity in vitro with a specificity gels, however, and migrates with an apparent size of 180 kDa identical to that observed for crude RNase E preparations. In (16). It has not been proven that the polypeptide encoded by addition, both UV crosslinking and RNA-protein blotting the ams/rne/hmpl gene is, in fact, a ribonuclease or is unambiguously showed that the Ams/Rne/Hmpl polypeptide intimately associated with the cleavage ofRNA either in vitro has a high affnity for RNA. Our results demonstrate that or in vivo. Indeed, it has been suggested that the gene product RNase E activity is directly attributable to, and is an inherent is not a ribonuclease but rather a regulatory protein or an property of, an RNA-binding protein, the ams/rne/hmpl gene auxiliary factor (8). It has also been proposed that RNase E product. activity requires the association of an unidentified catalytic subunit with other proteins, which could include the heat Regulating the balance between mRNA synthesis and decay shock protein GroEL (17). is a significant means of controlling gene expression. In the In an effort to understand the role ofRNase E with respect bacterium Escherichia coli, mRNA decay is usually initiated to substrate recognition and RNA processing, we have over- by endonucleolytic cleavage and followed by exonucleolytic expressed and purified the ams/rne/hmpl gene product to degradation at the new 3' ends by polynucleotide phospho- near homogeneity and have assayed its activity on various rylase and ribonuclease II (1). The endoribonuclease RNase substrate RNAs. In addition, we have utilized UV crosslink- E, which processes pre-5S rRNA from a larger precursor, 9S ing and RNA-protein blots to assess the binding affinity of RNA (2), has been demonstrated both genetically and bio- the full-length or truncated protein. Our results demonstrate chemically to be necessary for the decay of many specific that the ams/rne/hmpl gene product, to which we will refer mRNAs (for reviews, see refs. 3 and 4). The discovery that as Ams/Rne/Hmpl, has a high affinity for RNA and is an essential gene required for RNase E activity, rne, is allelic independently capable ofcorrectly processing several known to ams (altered mRNA stability) (5-8), manifested by ams-1, substrates of RNase E, including the 9S precursor to pre-5S a temperature-sensitive mutation responsible for a 6-fold rRNA. increase in the chemical half-life of pulse-labeled mRNA under nonpermissive conditions (9), constitutes strong evi- MATERIALS AND METHODS dence that RNase E is the rate-limiting factor in the turnover of many mRNAs. The site of cleavage by RNase E occurs Bacterial Strains and Plasmids. The E. coli strain 18-11BP A A (18) was obtained from M. P. Deutscher (University of within the consensus sequence GAUUU (3), which has been Connecticut Health Center, Farmington). The vector pET-11 found to be single stranded and flanked by stem-loop struc- and its host strain for expression, E. coli BL21(DE3) (19), tures (3, 10, 11). In vivo experiments have demonstrated that were obtained from Novagen. The recombinant bacterio- a substrate for RNase E, RNA I of the ColEl plasmid, phage A234 (20), originally AE3G11 (21) and the plasmid requires several unpaired nucleotides at its 5' terminus for pFMK33 (20), both containing all or part of the ams/rne/ efficient cleavage and suggest that RNase E may be sensitive hmpl gene, were obtained from D. Steege (Duke University, to 5'-terminal base pairing (12). Durham, NC) and S. R. Kushner (University of Georgia, Studies of the biochemical properties of the enzyme have Athens), respectively. pFMK33 was digested with Xmn I and generated conflicting data. RNase E was originally thought to BamHI. The 2.7-kbp fragment spanning residues 329-3068 of be a 70-kDa protein that could be rendered temperature the ams/rne/hmpl sequence (coordinates of ref. 16) was sensitive by the rne-3071 mutation (13). Genes capable of ligated into the unique BamHI site of pET-li. Plasmid complementing the ams-) or rne-3071 mutations have been pGM101 contained the 2.7-kbp fragment correctly oriented cloned, but small errors in sequence determination led to the relative to the vector, with the blunt end at the Xmn I site prediction of the ams/rne gene product being a polypeptide ligated directly to the BamHI site of the vector and the ofeither91 kDaor62 kDa, respectively (14, 15). The ams/rne four-residue gap on one strand repaired. To reconstruct the gene was recently recloned and sequenced as hmpl, a gene complete ams/rne/hmpl gene, a fragment of DNA spanning The publication costs ofthis article were defrayed in part by page charge Abbreviations: IPTG, isopropyl ,B-D-thiogalactopyranoside; DTT, payment. This article must therefore be hereby marked "advertisement" dithiothreitol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9006 Downloaded by guest on October 2, 2021 Biochemistry: Cormack et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9007 residues 3008-3824 was prepared by PCR amplification ofthe followed by the addition of an equal volume of SDS sample corresponding region of A234 with oligonucleotide primers buffer. The samples were separated in an SDS/7% polyacryl- spanning residues 3008-3031 (5'-AGTCGCCAATGCCGT- amide gel, stained with Coomassie brilliant blue R-250, and TGACCGTAG) and complementary to residues 3801-3824, exposed to x-ray film. with a C -- G mismatch at position 3811 to form a new BamHI RNA-Protein Blots. SDS/7% PAGE gels were used to site (5'-GGTTAGCAAGGATCCCATTCGATG). The prod- separate proteins prepared by boiling the equivalent of 50 p1 uct of amplification was digested with BamHI, purified, and of midlogarithmic phase cultures in SDS sample buffer. The ligated into pGM101 cleaved with BamHI to form pGM102, proteins were electroblotted to a nitrocellulose membrane which contained the complete ams/rne/hmpl gene (see Fig. using a carbonate transfer buffer (24) for 1-3 hr at a constant 1). The orientation and complete nucleotide sequence of the current of 250 mA. After transfer, the blot was placed in 750-bp BamHI fragment in pGM102 were checked by restric- TEN50 buffer (10 mM Tris-HCl, pH 8.0/1 mM EDTA/50 mM tion mapping and by DNA sequencing. Strain BL21(DE3) NaCI) and stored at 4°C for a minimum of 16 hr. Marker lanes was transformed with pGM101, pGM102, and pET-li to form were stained with 0.2% Ponceau S in 3% trichloroacetic acid. strains GM400, GM402, and GM403, respectively. Individual strips of the blot were incubated in binding buffer Preparation of Ams/Rne/Hmpl. Appropriate cultures [TEN50/0.02% Ficoll 400/0.02% polyvinylpyrrolidone/ were grown in a rich medium (19) at 28°C to early logarithmic 0.02% bovine serum albumin (fraction V)/yeast RNA (250 phase and induced with 0.5 mM isopropyl f-D-thiogalacto- pg/ml)] for 90 min at 44°C. 32P-labeled probe RNA generated pyranoside (IPTG). Cultures were harvested 5 hr later, and from in vitro transcription (specific activity 1.5 x 108 the supernatant from a 30,000 x g centrifugation was (S-30) was added x 105 and were precipitated with 26% (wt/vol) ammonium sulfate to yield the cpm/pug) (5 cpm/ml), the blots incubated for 90 min at 44°C. The blots were washed at AS-26 fraction as described (22) and dialyzed for 5 hr. ambient temperature for 10 min in TEN50 buffer, 10 min in Samples containing 2.5-3.0 mg of protein were heated for 5 min at 100°C in SDS sample buffer [60 mM Tris-HCl, pH TEN50 with 200 mM NaCl, and then for 10 min in TENS0 6.8/1.5% (wt/vol) SDS/50 mM dithiothreitol (DTT)/5% (wt/ with 500 mM NaCl. The blots were dried and exposed to x-ray vol) glycerol], applied to a 100 mm x 1.5 mm x 4 mm well film. in an SDS/5.5% polyacrylamide (49:1, acrylamide to N,N'- methylenebisacrylamide) gel, and separated by electropho- RESULTS resis. The outer edges of the gel were stained and used as a guide to excise the band containing the overexpressed poly- Overexpression and Purification of Ams/Rne/Hmpl. The peptide, which was subsequently eluted from the gel slice structure of a plasmid containing most ofthe ams/rne/hmpl using the Bio-Rad model 422 electroeluter.

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