Biochemistry 1990, 29, 9249-9256 Takeda, Y.,Folkmanis, A,, & Echols, H. (1977) J. Biol. Chem. 127,6177. Weber, P. L., Wemmer, D. E., & Reid, B. R. (1985) Biochemistry 24, 4553.
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Wells, J. A., Vasser, M., & Powers, D. B. (1985) Gene 34, 315. Wilkins, M. H. F. (1956) Coldspring Harbor Symp. Quant. Biol. 21, 75.
micF RNA Binds to the 5’ End of ompF mRNA and to a Protein from Escherichia coli? Janet Andersent and Nicholas Delihas* Department of Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 1I794 Received March 30, 1990; Revised Manuscript Received June 20, I990
F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF m R N A (Andersen et al., 1989). A 93-nucleotide micF R N A was synthesized in vitro directly from polymerase chain reaction generated D N A which was designed to contain a functional T7 R N A polymerase promoter upstream of the micF R N A gene and an appropriate restriction site for transcription termination. A transcript ( 1 50 nucleotides) containing the ribosomal binding domain of ompF m R N A messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector. A stable duplex was formed between micF R N A and the 150-nucleotide 5’ transcript of ompF m R N A after incubation at 37 O C in a physiological buffer. The melting curve of the duplex formed by micF R N A and 150-nucleotide transcript revealed a T, of 56 “C and a AT,,, that spans about 20 “ C ; both are consistent with the proposed structure for the micF/ompF duplex. In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E . coli protein was found to bind specifically to micF R N A . The protein also bound weakly to the 150-nucleotide ompF transcript. The data are the first to demonstrate the complex between micF R N A and the 5’ end of ompF m R N A and suggest that in vivo a micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF m R N A during thermoregulation of OmpF porin. ABSTRACT: micF R N A regulates the levels of outer membrane protein
%e outer membrane protein OmpF, a major porin protein of Escherichia coli, is regulated in response to changes in temperature, osmolarity, and other stress conditions during growth [Lugtenberg et al., 1976; Hall & Silhavy, 1981; Andersen et al., 1989; see Forst and Inouye (1988) for a review]. It has been shown that chromosomally derived 4.5s micF RNA plays an essential role in the thermal regulation of OmpF (Andersen et al., 1989). The 93-nucleotide micF RNA represses OmpF synthesis by decreasing the levels of ompF mRNA (1.1 kb) in response to temperature increase as well as to the other stress-related factors (Andersen et al., 1989). While micF RNA is necessary for the observed decreased levels of ompF MRNA, it is not sufficient; another factor, possibly a cognate protein, was deduced to participate with micF RNA in the regulation of the messenger’s levels (Andersen et al., 1989). Although the micF RNA gene is at 48’ on the E . coli chromosome and distal from the ompF gene at 21’ (Inokuchi et al., 1982; Mizuno et al., 1983; Bachmann, 1987), micF RNA is believed to function as a natural antisense RNA against the mRNA for OmpF. Consistent with its hypothesized role as an antisense RNA, the suppression of OmpF via micF RNA has been shown to occur at a posttranscriptional level (Misra & Reeves, 1987; Cohen et al., 1988), and This work was supported by National Science Foundation Grant DBM 88-03122 (N.D.). *To whom correspondence should be addressed. *Present address: Department of Pathology, School of Medicine, SUNY Stony Brook, Stony Brook, N Y 11794-8691.
0006-2960/90/0429-9249$02.50/0
micF RNA can significantly inhibit translation of ompF mRNA if overexpressed (Mizuno et al., 1983, 1984; Andersen et al., 1989). A model of micF RNAlompF mRNA has been proposed (see Figure 1). In this model, the primary sequence of micF RNA shows extensive complementarity with the 5’ end of ompF mRNA in and around its ribosome binding domain; however, the complementarity is imperfect since there are several looped-out positions and non-Watson-Crick base pairing in the proposed duplex (Mizuno et al., 1984; Andersen et al., 1987). Since micF RNA is found in low levels in the cell (Andersen et al., 1987), it is difficult to obtain in sufficient quantities for in vitro studies. In a novel approach, polymerase chain reaction (PCR) was used to generate DNA from which bona fide micF RNA could be synthesized by T7 RNA polymerase. Using T7-synthesized RNAs, we show that micF RNA hybridizes to the 5’ end of ompF mRNA and forms a stable duplex. We also show the micF RNA binds specifically to a protein from E . coli, suggesting that a micF R N P particle may regulate the stability of ompF mRNA.
EXPERIMENTAL PROCEDURES Polymerase Chain Reaction Generated DNA. The PCR technique (Mullis & Faloona, 1987) was used to introduce a T7 RNA polymerase promoter upstream of the micF RNA gene and to create a restriction site for run-off transcription at the end of the gene (Figure 2A). Due to paucity of restriction sites around the micF RNA gene, this novel method was chosen in lieu of conventional cloning to position the
0 1990 American Chemical Society
Andersen and Delihas
9250 Biochemistry, Vol. 29, No. 39, 1990
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FIGURE 1: Proposed secondary structural model of the duplex formed by micF RNA and ompF mRNA around the region of the initiation codon and Shine-Dalgarno sequence on the messenger (Mizuno et al., 1984; Andersen et al., 1989). The top strand shows the complete sequence of micF RNA, and the bottom strand shows the sequence of the 5’ end of ompF mRNA from positions 62 to 121.
promoter upstream of the gene and create a site for the run-off transcription so that bona fide micF RNA could be synthesized by T7 RNA polymerase in vitro. A similar PCR strategy was employed to obtain sufficient transcripts for reverse transcription reactions in a technique called genomic amplification with transcript sequencing (GAWTS) (Stoflet et al., 1988). As in GAWTS, it was not necessary to clone the resulting PCR DNA in order to transcribe its micF RNA gene with T7 RNA polymerase. A 300 bp restriction fragment carrying the micF RNA gene (the CX28 fragment; Mizuno et al., 1984) was cut from plasmid pAM336 (Andersen et al., 1987) with XbaI restriction endonuclease, and the DNA fragment was gel-purified on low melting point (LMP) agarose. The DNA was extracted from the agarose using Elutip-D columns according to the direction from the manufacturer (Schleicher & Schuell). Two oligonucleotides were designed to be used in the PCR reaction along with the gel-purified 300 bp restriction fragment carrying the micF RNA gene. Two restriction sites (EcoRI and HindIII) were built into the DNA sequence as potential cloning sites. Also, at the 5’ ends of each oligonucleotide, two extra nucleotides were added beyond the restriction site sequence in an effort to avoid interference with restriction cutting of the PCR DNA which might result from “breathing” at the ends of the double-stranded DNA. The sequence of the 5’ oligonucleotide (51-mer) is 5’-
GCgaattcCGAAATTAATACGACTCACTATAGGCTATCATCATTAACTTTA-3’. It contains (5’ to 3’) an EcoRI sequence (lower case), the consensus T7 RNA polymerase promoter sequence (underlined) plus 6 upstream nucleotides (Ikeda & Richardson, 1986), an extra G residue to assure accurate transcription initiation, and the sequence of the first 19 nucleotides from the noncoding strand [(+) strand] of micF gene sequence (boldface letters). The sequence of the 3’ oligonucleotide (36-mer) is 5’ATaagctttaaaAAAAACCGAATGCGAGGCATCCGG-3’.It contains (5’ to 3’) overlapping HindIII and DraI sequences in antisense orientation (lower case) followed by 17 nucleotides complementary to the noncoding strand [(+) strand] at the 3’ end of the micF gene (boldface letters). The DraI sequence also overlaps A residues that appear in the micF sequence. Cutting the PCR-generated DNA with DraI, a blunt-end cutter, results in run-off transcription at the last A/T base pair of the micF gene. Since cloning of the PCR product is not necessary in order to use the template DNA in in vitro transcription reactions, the sequence of the 3’ oligonucleotide may be abbreviated to only those sequences complementary to the noncoding strand. PCR T7 template has been made that synthesizes correct-size U6 snRNA using an abbreviated 3’
oligonucleotide (Andersen and Zieve, unpublished results). The 5’ and 3’ oligonucleotides were made on an Applied Biosystems DNA synthesizer, Model 380B, processed according to the manufacturer’s directions and gel-purified on a thin sequencing-type polyacrylamide gel containing 50 mM Tris-borate, pH 8.3, 1 mM EDTA (TBE), and 7 M urea. Purified oligomer concentration was determined from readings using an estimated extinction coefficient of 25 pg/mL for A260 = 1. The PCR reaction was done according to the instructions from the Perkin Elmer Cetus GeneAmp DNA amplification kit on a PCR machine from the same company, and all reagents used in the reaction, other than the restriction fragment and the oligonucleotides, came from the purchased kit. The appropriate annealing temperature was estimated from the number of G/C (4 OC per bp) and A/T (2 OC per bp) base pairs formed between the oligomers and the CX28 restriction fragment, and a temperature about 5 OC less than the estimated lowest value was chosen as the annealing temperature for the reactions, in this case, 43 OC. Default parameters of Perkin-Elmer Cetus program 3 were used for the chain reaction except the program was modified to deliver an annealing temperature of 43 “C and, due to the small size of the expected product ( 130 bp), to allow polymerization for only 30 s. One nanogram of the CX28 restriction fragment was sufficient to obtain about 4-5 pg of finished product under these conditions. ompF Gene Cloned into T7 Expression Vector. The ompF gene was cloned into a T7 expression vector by conventional cloning techniques (Maniatis, 1982) as outlined in Figure 2B. Plasmid pGR201 (Ramakrishnan et al., 1985) was first cut with EcoRI and AuaI restriction endonucleases. After phenol/chloroform extraction and ethanol precipitation, the cut DNA was subjected to Klenow treatment so that BamHI linkers (8-men) could be ligated to the resulting blunt ends. The ligated linkers were subsequently restricted with BamHI endonuclease and kinased with T4 polynucleotide kinase and ATP before the DNA was purified by electrophoresis on LMP agarose. A 2.6-kb restriction fragment was extracted from the gel by using Elutip-D columns and subsequently ligated into the prepared T7 expression vector, pET7 (Rosenberg et al., 1987). Plasmid pET7 had been prepared by restriction with BamHI, treatment with bacterial alkaline phosphatase, and gel purification. Ligation occurred overnight at 14 OC using T4 DNA ligase. The resulting plasmid, pJANO10, was subsequently cut with StuI and XbaI blunt-end restriction endonucleases, and gelpurified. Religation of the large restriction fragment resulted in a second plasmid, pJANO11, which has a functional T7 promoter and two initiating G residues (from the StuI cut) N
Biochemistry, Vol. 29, No. 39, 1990 9251
micF RNA Binds to ompF mRNA and a Protein A. *E
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