JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4578-4586 0021-9193/91/154578-09$02.00/0

Vol. 173, No. 15

Copyright © 1991, American Society for Microbiology

Structure and Function of a Bacterial mRNA Stabilizer: Analysis of the 5' Untranslated Region of ompA mRNA LI-HOW CHEN, SHERI A. EMORY, ANGELA L. BRICKER, PHILIPPE BOUVET, AND JOEL G. BELASCO* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 11 March 1991/Accepted 22 May 1991

The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serraia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants.

Degradation of mRNA is a cellular process that directly affects gene expression through its impact on mRNA concentration. The lifetimes of individual messages within a single cell can differ by as much as 3 orders of magnitude and are often modulated in response to changes in a cell's growth state or environment. In Escherichia coli, for example, mRNA half-lives range from several seconds to nearly 1 h, with an average lifetime of about 2 to 4 min (9, 10, 43). Despite the importance of mRNA degradation to gene regulation, the molecular basis for these marked differences in mRNA stability is just beginning to be understood (2, 6, 23). A key to explaining these differences in decay rate is to identify the structural features of long- or short-lived messages that make them especially resistant or susceptible to degradation in vivo. Among the most stable E. coli messages is the transcript of the ompA gene, which encodes a major outer membrane protein (OmpA). In cells growing rapidly at 30°C, the halflife of this message is about 17 min (10, 47). Regulation of OmpA protein synthesis by the rate of cell growth is achieved primarily through modulation of the stability of ompA mRNA, whose half-life can fall by as much as a factor of 4 in slowly growing cells (10, 25, 39). The remarkably long lifetime of the E. coli ompA transcript is not due to some unusual feature of its 3'-terminal stem-loop structure (3), which, like the 3' hairpins of other bacterial messages, is essential for protecting against 3'exonuclease digestion in vivo (8, 34, 38). Instead, multiple lines of evidence indicate that the extraordinary longevity of the ompA message is attributable to its long (133-nucleotide [nt]) 5' untranslated region (UTR). This RNA segment functions in E. coli as a growth rate-regulated mRNA stabilizer (3, 10). Thus, deletion of the terminal 115-nt ompA mRNA segment that lies upstream of the ribosome binding site reduces the half-life of the ompA transcript to just 3 to 4 min, that of an ordinary E. coli message (10). In addition, substitution of the 5' UTR and first few codons of ompA mRNA for the corresponding portion of the labile bla gene transcripts increases the half-life of bla mRNA from 3 min to *

Corresponding author. 4578

17 to 18 min (3, 10). Other labile E. coli messages also can be stabilized by fusion to the ompA 5' UTR (16), suggesting that many mRNAs are degraded via a common pathway against which the ompA 5' UTR provides protection. As observed for the wild-type ompA transcript, stabilization of heterologous mRNA by the E. coli ompA 5' UTR is growth rate dependent (10). The mechanism by which the ompA 5' UTR stabilizes mRNA is not an indirect consequence of close spacing of translating ribosomes (i.e., of unusually frequent translation initiation), which in theory might sterically hinder RNase attack (10, 16, 25). Instead, this RNA segment, either alone or in concert with an associated protein, appears to function directly to protect the ompA transcript from cleavage by an RNase that is sensitive to structural features near the 5' end of mRNA. Unlike three other 5' RNA segments that have been shown to prolong mRNA lifetimes in bacteria (1, 13, 44), message stabilization by the ompA 5' UTR occurs under normal conditions of rapid cell growth and does not require phage infection or treatment with an inhibitor of translation. As an essential step toward understanding the mechanism of mRNA stabilization by the ompA 5' UTR, we report here its secondary structure, as determined by phylogenetic analysis, in vitro probing with a structure-specific RNase, and in vivo methylation by dimethylsulfate. Our studies reveal that despite extensive sequence divergence, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes display striking structural similarities. Moreover, each of these 5' UTRs functions as an effective mRNA stabilizer in E. coli. These findings provide a structural foundation for clarifying the molecular basis of differential mRNA stability in E. coli. MATERIALS AND METHODS Strains and plasmids. E. coli K-12 strains C600S (40) and PM191 (29) are both derivatives of strain C600. E. coli SE600 is identical to C600S except for a deletion of the chromosomal ompA gene (10). Plasmids pOMPA102 and pOMPA103 were constructed in two steps. First, a 3.1-kb XmnI fragment of pTU100 (7) carrying the entire wild-type E. coli ompA gene was cloned

VOL. 173, 1991

with EcoRI linkers into the EcoRI site of a derivative of pPM30 (29) whose AccI site had been eliminated. In the resulting plasmid, pOMPA100, the direction of transcription of the ompA gene is the same as that of the bla gene present on the same plasmid. Replacement in pOMPA100 of a 0.14-kb AccI-FokI fragment encoding the E. coli ompA 5' UTR with the corresponding AccI-FokI fragment of pTU5Se (5), a plasmid that carries the S. marcescens ompA gene, generated pOMPA102. Plasmid pOMPA102 encodes a hybrid ompA transcript comprising the Serratia ompA 5' UTR fused to the coding region and 3' UTR of the E. coli ompA transcript. The junction of this gene fusion is precise except for an additional pair of adenosine residues introduced immediately upstream of the translation initiation codon. These two extra nucleotides are present at this position in the wild-type E. coli ompA message but not in that of S. marcescens. Replacement of the same AccI-FokI fragment of pOMPA100 with the corresponding fragment of pTU7En (4), a plasmid that carries the Enterobacter aerogenes ompA gene, generated pOMPA103. Plasmid pOMPA103 encodes a hybrid ompA transcript comprising a perfect fusion of the Enterobacter ompA 5' UTR to the coding region and 3' UTR of the E. coli ompA transcript. Plasmid pSOB2 encodes a hybrid gene (sob2) in which the sixth codon of the Serratia ompA gene is fused in-frame to codon 23 of the bla gene of pJB322 (3), thereby reconstituting a BstEII site at the fusion junction. This plasmid encodes a cytoplasmic form of ,B-lactamase. It was constructed from plasmid pOB1, which is a pOB2 (3) variant that encodes a complete pre-OmpA signal peptide. First, a 0.33-kb AccIBstEII (filled-in) pOBi fragment containing the promoterproximal portion of the obi gene was replaced with a 0.16-kb AccI-NruI pOMPA102 fragment containing the promoterproximal portion of the ompA102 gene. Substitution of the 0.75-kb Hinfl (filled in and adapted with an EcoRI linker)PstI fragment of the resulting plasmid for the 1.45-kb EcoRIPstI fragment of pOMPA+4 (10) generated pSOB2. Plasmids pT7ompA+5 and pT7ompA+5* were constructed from pSE18, a derivative of pT7/T3oa-18 (Bethesda Research Laboratories) with a deletion of bp 2 through 24 downstream of the T7 RNA polymerase transcription initiation site (bp 1). Insertion of a 0.15-kb BclI-NruI fragment of pOMPA+4 (10) between the BamHI and Sall (filled-in) sites of pSE18 generated pT7ompA+5 and recreated the Sall site. Introduction of a symmetrical DNA linker (5'-GCCC ACCGGCAGCTGCCGGTGGGC-3') at the HincIl site of pT7ompA+5 generated pT7ompA+5*. Analysis of RNA secondary structure. Phylogenetic determination of RNA secondary structure was performed as described previously (22). RNA for polynucleotide phosphorylase (PNPase) digestion was synthesized and uniformly radiolabeled in vitro, using T7 RNA polymerase and either pT7ompA+5 DNA linearized with HinclI or pT7ompA+5* DNA linearized with HindIll (24). RNA synthesis was stopped by addition of excess EDTA. After phenol-chloroform extraction, gel filtration through Sephadex G-50, and ethanol precipitation, RNA samples were redissolved in water and stored at -20°C. PNPase digestion was performed at 37°C in a reaction mixture (50 RI) containing Tris Cl (50 mM, pH 8.0), sodium chloride (60 mM), magnesium chloride (5 mM), potassium phosphate (10 mM), labeled RNA (4 x 104 cpm), and PNPase (0.075 U; from Micrococcus luteus [Boehringer]). Samples (5 pul) were quenched with EDTA at time intervals and fractionated by electrophoresis on a 6% polyacrylamide-8.3 M urea gel.

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Methylation of RNA by dimethylsulfate in E. coli and in vitro and mapping of methylation sites was performed as described previously (28, 32). Bacteria for RNA methylation (C600S, SE600/pOMPA102, or SE600/pOMPA103) were grown and harvested under the conditions used for measurements of mRNA stability in rapidly growing cells (10). For in vivo methylation, cells growing in liquid culture were treated with 0.1% (vol/vol) dimethylsulfate for 5 min at 30°C. In vitro methylation of purified total cellular RNA was performed with 0.3% (vol/vol) dimethylsulfate for 10 min at 30°C in a buffer containing sodium cacodylate (80 mM, pH 7.2), potassium chloride (150 mM), and magnesium chloride (25 mM). A 5'-end-labeled DNA oligonucleotide (5'-AGCGA AACCAGCCAGTGCCACTGC-3') was used as a primer to map methylation sites within the ompA 5' UTR. Annealing of this primer to an mRNA sequence 22 to 45 nt downstream of the ompA translation initiation codon was accomplished by heating the primer (0.1 pmol) and total cellular RNA (5 pug) to 90°C in a buffer (7.5 ,ul) containing N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; 50 mM, pH 7.0) and potassium chloride (100 mM) and then cooling the mixture slowly to 45°C. The primer was then extended at 42°C with avian myeloblastosis virus (AMV) reverse transcriptase (0.32 U/pul; Boehringer) in a buffer (12.5 pul) containing Tris Cl (50 mM, pH 8.0), HEPES (30 mM, pH 7.0), potassium chloride (60 mM), magnesium chloride (10 mM), dithiothreitol (10 mM), and four deoxynucleoside triphosphates (0.2 mM each). Sites of premature termination by a recombinant form of Moloney murine leukemia virus (MuLV) reverse transcriptase that lacks 48 carboxy-terminal amino acids (0.32 U/,u; New England BioLabs) were mapped on unmethylated ompA mRNA at 42°C under the same conditions. Measurement of mRNA lifetimes. Bacterial culture, RNA extraction, S1 analysis, and calculation of mRNA half-lives were performed as previously described (10). Cells were grown at 30°C in supplemented LB medium (10) to achieve rapid growth or in morpholinepropanesulfonic acid (MOPS)acetate medium (37) to achieve slow growth. The probe for detecting ompA102, ompA103, and wild-type ompA mRNA was a 0.6-kb BstEII-EcoRI fragment of pOMPA+4 5' labeled at the BstEII end (10). The sob2 transcripts were detected with a 1.0-kb Hinfl-EcoRI fragment of pBLA200 5' labeled at the Hinfl end (10). RESULTS Secondary structure of the ompA 5' UTR. Phylogenetic comparison has proven to be a powerful and reliable method for deducing RNA secondary structure from sequence data (12, 21, 22, 42). The principle underlying this analytical method is that for a given RNA molecule, the base-pairing potential of functionally important double-helical regions generally is maintained during evolution despite sequence divergence. Thus, evidence for the existence in vivo of a proposed RNA duplex is provided by compensatory sequence changes that preserve the base-pairing potential of this structure among homologous RNA molecules in a number of organisms. On the other hand, if related RNA molecules in different species cannot all fold to form a particular duplex structure, then this structure either does not exist in vivo or is functionally unimportant. DNA sequences previously have been determined for the ompA genes of five different enteric bacteria: E. coli, Salmonella typhimurium, Shigella dysenteriae, Enterobacter aerogenes, and S. marcescens (4, 5). Among these genes,

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Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA.

The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary s...
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