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J. Biol. Chem., Vol. 277, Issue 9, 7239-7245, March 1, 2002

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The Nucleic Acid Melting Activity of Escherichia coli CspE Is Critical for Transcription Antitermination and Cold Acclimation of Cells^*

Sangita Phadtare, Masayori Inouye^§, and Konstantin Severinov^¶

From the  Department of Biochemistry, Robert Wood Johnson Medical School and the ^¶ Waksman Institute, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854

Received for publication, December 3, 2001, and in revised form, December 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of bacterial Csp (cold-shock protein) family promote cellular adaptation to low temperature and participate in many other aspects of gene expression regulation through mechanisms that are not yet fully elucidated. Csp proteins interact with single-stranded nucleic acids and destabilize nucleic acid secondary structures. Some Csp proteins also act as transcription antiterminators in vivo and in vitro. Here, we selected a mutation in the cloned cspE gene that abolished CspE-induced transcription antitermination. In vitro, mutant CspE showed RNA binding activity similar to that of the wild-type CspE but was unable to destabilize nucleic acid secondary structures. Thus, nucleic acid melting ability of CspE and its transcription antitermination activity are correlated. In vivo, mutant cspE was functional with respect to up-regulation of expression of rpoS, but, unlike the wild-type cspE, it did not complement the cold-sensitive phenotype of the quadruple cspAcspBcspGcspE deletion strain. Thus, the nucleic acid-melting activity of Csp is critical for its prototypical function of supporting low temperature survival of the cell.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When an exponentially growing culture of Escherichia coli is shifted from 30 to 15 °C, the cells exhibit a cold-shock response (1). This response is characterized by a transient arrest of cell growth during which a number of genes are induced, in contrast to a severe inhibition of general protein synthesis. Among the cold-shock proteins, the most prominent is CspA.¹ Other cold-inducible proteins include transcription factor NusA (2), polynucleotide phosphorylase (3), initiation factor IF2 (4), RecA (5), histone-like protein H-NS (6), DNA gyrase (7), ribosome-associated factors RbfA (8), and CsdA (9).

In E. coli, there are nine Csp proteins, from CspA through CspI, of which CspA, CspB, CspG, and CspI are cold-shock-inducible. CspC and CspE are constitutively produced at 37 °C, whereas CspD is induced upon nutritional deprivation. The induction patterns of CspF and CspH are not known (for review see Refs. 10 and 11). CspA homologues are widely distributed in prokaryotes, and CspA is homologous to the cold-shock domain in human Y-box protein YB-1 and the Y-box proteins from other eukaryotes (for review see Refs. 12 and 13).

None of the CspA homologues appear to be singularly responsible for cold-shock adaptation, because deletions in any one of the csp genes do not result in cold sensitivity. The double and triple deletion mutations in E. coli csps (cspAcspB, cspAcspG, cspBcspG, cspAcspI, or cspAcspBcspG) did not result in cold sensitivity (14). In a triple deletion strain of cspAcspBcspG, CspE accumulated at low temperatures, suggesting that members of the CspA family may functionally substitute for each other during cold acclimation of cells (14).

In addition to their apparent, but poorly understood role during cold-shock response, many cellular processes appear to respond to changes in Csp protein concentrations even at higher temperature. In E. coli, the presence of camphor leads to chromosome decondensation. Overproduction of CspE led to camphor resistance and chromosome condensation (15). CspC and CspE were also shown to be involved in the regulation of the expression of rpoS, a gene encoding a global stress response regulator, and uspA, a gene encoding a protein that is induced by numerous stresses (16).

In vitro, CspA binds to RNA with a low sequence specificity and a low binding affinity (17). CspB, CspC, and CspE are able to more selectively bind RNA and single-strand DNA (18). Binding of CspA to RNA destabilizes RNA secondary structures and, hence, it presumably facilitates translation at low temperatures (17). Hanna and Liu (19) demonstrated the interaction between CspE with the nascent RNA in transcription elongation complexes, suggesting that this protein is involved in transcription regulation. They also found that purified CspE interfered with Q-mediated transcription antitermination. It has also been shown that CspA, CspE, and CspC decreased transcription termination at several intrinsic terminators and also affected transcription pausing (20). Recently, it was found that purified CspE impedes poly(A)-mediated 3'- to 5'-exonucleolytic decay by polynucleotide phosphorylase by interfering with its digestion through the poly(A) tail and inhibits both internal cleavage and poly(A) tail removal by RNase E (21).

The relevance, if any, of all of these biochemical functions of CspA homologues to their physiological function is not known. In the present paper, we explored the physiological relevance of transcription antitermination activity of CspE by mutational analysis of a cloned cspE gene. We show that a mutation of an evolutionarily conserved histidine residue, His³², which resides within RNP2, a putative RNA-binding site of CspE, abolishes transcription antitermination in vivo and in vitro. The mutation leaves the RNA-binding activity of CspE unaffected but abolishes the nucleic acid-melting activity of CspE. The mutant CspE overexpression does not complement the cold sensitivity of the cspAcspBcspGcspE quadruple deletion but is able to up-regulate rpoS expression in the stationary phase. Thus, our results provide further evidence that (i) CspE, like other known transcription antitermination factors, targets the secondary structure elements in the nascent RNA, and (ii) transcription antitermination activity of CspE is correlated with its prototypical in vivo function during cold-shock acclimation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains-- E. coli wild-type strain JM83 (22), its quadruple deletion strain of cspAcspBcspGcspE (14), and the strain RL211 (23) were used in this study. The bacterial cultures were grown in Luria broth (LB). Antibiotics such as ampicillin (50 µg ml¹) or chloramphenicol (30 µg ml¹) were supplemented as required.

Random Mutagenesis-- The cspE gene was randomly mutagenized using the methods of Lerner et al. (24) and Spee et al. (25). Each of the four NTPs (ATP, CTP, GTP, and TTP) was depleted one at a time in PCR. MnCl₂ (0.5 mM) and dITP (200 µM) were also included in the reaction, and Taq polymerase was used. The PCR products corresponding to the mutagenized open reading frame of cspE were cloned in pINIII plasmid, using NdeI and BamHI, and transformed in the strain RL211. The clones were screened for their sensitivity to chloramphenicol. The in vivo assay for transcription antitermination using this strain is described previously (20). The plasmids were isolated from the colonies showing growth on LB plates containing ampicillin but not on the plates containing both ampicillin and chloramphenicol. The plasmids were retransformed in the strain RL211, and sensitivity of the transformants to chloramphenicol was confirmed. The plasmid (pINcspE-N7K-H32R) consistently showing sensitivity to chloramphenicol was sequenced, and the insert corresponding to open reading frame of each cspE mutant was subcloned in pET11a vector to yield the plasmid pET11a-cspE- N7K-H32R. This vector contains a T7 promoter under the control of the lac operator.

Site-directed Mutagenesis-- The single amino acid point mutations within the cspE coding region were introduced through site-directed mutagenesis using PCR reaction. The resultant DNA fragments were cloned in pINIII and pET11a vectors. The plasmids (pINIIIcspE-N7K, pINIIIcspE-H32R, pET11a-cspE-N7K, and pET11a-cspE-H32R) were sequenced to confirm the mutant sequences.

Expression and Purification of the Proteins-- In vivo expression of CspE and its mutants was examined using corresponding isopropyl -D-thiogalactopyranoside (IPTG)-inducible pINIII expression vectors as described previously (16, 20). The cells were grown at 37 °C to A₆₀₀ of 0.5, and the CspE expression was induced with 1 mM IPTG for 60 min at 37 °C, followed by analysis with SDS-PAGE. CspE and its mutant proteins were purified by Q-Sepharose and hydroxyapatite column chromatography as described previously (20).

Circular Dichroism Studies-- CD measurements were performed on an automated AVIV 60DS spectrophotometer fitted with a thermostatted cell holder that is controlled by an on-line temperature control unit. Quartz rectangular cells (Precision Cells, Hicksville, NY) with a path length 1 mm were used. These cells were maintained at 4 °C during the experiment. For CD studies, CspE and its mutant proteins in 20 mM potassium phosphate buffer, pH 7.0, were used. Solutions were filtered through 0.22-µm filters before use. In the thermal unfolding experiments, temperatures were increased from 10 to 90 °C in 1 °C intervals, with a 30-s equilibration at each temperature.

In Vitro Transcription-- The in vitro transcription was carried out as described previously (20). A DNA template containing the T7A1 promoter fused to the tR2 terminator was used. The elongation complexes stalled at position +20 (EC²⁰) were prepared in transcription buffer (40 mM KCl, 40 mM Tris-HCl, pH 7.9, and 10 mM MgCl₂) containing Ni²⁺-nitriloacetic acid-agarose, 20 nM T7 A1 promoter-DNA fragments, 40 nM His-tagged RNA polymerase, and 0.5 mM ApU. Reaction mixtures were incubated at 37 °C for 15 min to form the open complexes and were then transferred to room temperature. 50 µM ATP, 50 µM GTP, and 2.5 µM [-³²P]CTP (300 Ci/mmol) were added. After 10-min incubation, the agarose beads were thoroughly washed with transcription buffer. Equal aliquots of purified EC²⁰ were then supplemented with Csp proteins (1.5 µg) and NTPs (250 µM), and the reactions were incubated at room temperature for 10 min. After the transcription reactions, 20 mM EDTA and 10 mg/ml heparin was added to the reaction mixtures to avoid nonspecific retardation of RNA in the gel. The reactions were terminated by formamide-containing loading buffer. The products were analyzed by urea-PAGE electrophoresis (7 M urea/10% polyacrylamide) followed by autoradiography and phosphorimaging analysis.

RNA Binding Assay-- The 88-nucleotide RNA fragment to which CspE binds preferentially as shown previously by SELEX (systematic evolution of ligands by exponential enrichment) was used, and the filter binding assays were carried out as described before (18). T7 RNA polymerase reaction was carried out in the presence of [-³²P]UTP to prepare the RNA probe. The RNA was then purified by phenol/chloroform and ethanol precipitation, and its integrity was checked by urea-acrylamide gel electrophoresis. RNA concentration was estimated by quantitating [-³²P]UTP incorporation.

Binding assay was carried out in a 15-µl reaction mixture containing binding buffer (10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, 10 mM KCl, 7.4% glycerol) and 50 fmol of RNA. After incubation on ice for 20 min, samples were analyzed by filter binding assays. Variable amounts of proteins were incubated with constant amounts (50 fmol) of RNA. The reaction mixtures were passed through nitrocellulose filters, which were washed thoroughly to remove unbound RNA. Radioactivity retained on filter was measured by liquid scintillation counter. About 1% of the input radioactivity was detected as background in the absence of any protein in the reaction mixture. This background was subtracted from the measured amounts to get specific binding values.

Western Blot-- The Western blot analysis was carried out as described previously (16). Wild-type cells containing pINIII, or the pINIII plasmids containing open reading frames of cspE or its mutants were grown at 37 °C in M9 medium. The exponentially growing cells at an A₆₀₀ of 0.5 were treated with 1 mM IPTG for 30 min and were then concentrated by centrifugation at 13,000 × g. The resulting cell pellets were suspended in SDS-loading buffer, and the proteins were resolved by SDS-PAGE. The Western blots were prepared according to the antibody manufacturer's protocol (Neoclone).

Molecular Beacons-- The molecular beacons were a gift from Dr. Sanjay Tyagi (New York University). The beacon used in this study was a 82-nucleotide-long hairpin-shaped molecule labeled with a fluorophore and quencher: tetramethyl rhodamine-AGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCACCGCCGACGACAGTCGCTAACCTCTCGCTAAGAACCCT-DABCYL. It was made by ligating the two oligos: 5'-half-tetramethyl rhodamine-AGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCAC and 3'-half-CGCCGACGACAGTCGCTAACCTCTCGCTCAAGAACCCT-DABCYL. As a splint for ligation the following oligonucleotide was used: 5'-TAGCGACTGTCGTCGGCGGTGCATAGGGAAGCACAGATAAAA-3'. The ligated full-length product was purified on a 10% acrylamide gel containing 7 M urea. The product was eluted in GE buffer and precipitated with ethanol.

Fluorescence measurements were performed on an LS-5B spectrofluorometer (PerkinElmer Life Sciences), using 1-cm path length QS cuvettes (Hellma). The temperature of the cuvette was controlled by a circulating bath. The excitation and emission wavelengths used were 555 and 575 nm, respectively. The fluorescence of a 100-µl solution of 32 nM molecular beacon dissolved in 20 mM Tris-HCl, pH 7.5, containing 1 mM MgCl₂ was monitored as CspE or its mutant proteins (1.5 µg) were added. The reactions were carried out at room temperature. To check the effect of degradation of CspE by trypsin (50 times excess), the reactions were carried out at 37 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of CspE Mutant Proteins Deficient in Transcription Antitermination-- Previously we had reported the use of E. coli strain RL211 designed by Landick et al. (23) to demonstrate in vivo transcription antitermination activity of Csp proteins (20). This strain contains the cat gene preceded by a strong -independent trpL terminator and is therefore sensitive to chloramphenicol. When transcription termination at trpL is reduced, the cat gene is expressed and the cells become resistant to chloramphenicol. Cells overproducing CspE from IPTG-inducible pINIII plasmid formed colonies on chloramphenicol-containing medium, indicating that CspE overproduction caused transcription antitermination at the trpL terminator in vivo (20). In the present study, we used this property of CspE to design a screening assay for antitermination-deficient mutants. The cspE gene was subjected to random PCR mutagenesis and recloned in pINIII. The mutant plasmid pool was next transformed in RL211 strains, and transformants were screened for their sensitivity to chloramphenicol as described under "Experimental Procedures." 20% of the clones were sensitive to chloramphenicol; however, all of them except for one did not show the CspE protein expression, indicating that the lack of protein itself was responsible for the sensitivity to chloramphenicol. The one transformant consistently exhibiting sensitivity to chloramphenicol showed a comparable level of protein expression with that of the wild-type CspE. Sequencing of the plasmid isolated from this transformant showed that the insert corresponding to the open reading frame of cspE had two-nucleotide differences as compared with the wild-type cspE sequence. The mutant cspE had a AAG (Lys) codon at position 7 and a CGC (Arg) codon at position 32, whereas wild-type cspE has AAC (Asn) and CAC (His) codons at these positions, respectively. This result indicates that the change from Asn to Lys at CspE position 7 and/or His to Arg at CspE position 32 is the cause of the Cm^S phenotype.

Site-directed mutagenesis was next used to separate the two point mutations to create corresponding single mutants, CspE-N7K and CspE-H32R. We then tested if the overproduction of CspE proteins carrying these mutations can cause transcription antitermination in vivo using the RL211 cells as described above. Aliquots of the RL711 cells transformed with plasmids overexpressing the wild-type CspE, the double mutant, or the single mutants were spotted on LB plates containing ampicillin and IPTG, in the presence or in the absence of chloramphenicol. Cells carrying the pINIII vector alone were used as control. As expected, all cells exhibited growth in the absence of chloramphenicol, however, only those overproducing wild-type CspE or the N7K mutant were able to grow in the presence of chloramphenicol (Fig. 1A). Fig. 1B shows SDS-PAGE analysis of the expression levels of these proteins. The induction levels of all the four proteins were similar; therefore, the effect on transcription antitermination appears to be due to the defect in the protein activity itself. We conclude that the change from His to Arg at CspE position 32 is the cause of the Cm^S phenotype.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Mutations in CspE affect transcription antitermination in vivo. A, RL211 E. coli cells containing a cat gene cassette positioned downstream of the trpL terminator (23) were transformed with pINIII vector alone as a control or with pINIII containing cloned cspE, cspE-N7K-H32R, cspE-N7K, or cspE-H32R and spotted on LB plates containing 50 µg/ml ampicillin, 1 mM IPTG, and with and without 30 µg/ml chloramphenicol. The results of overnight cell growth are presented. B, SDS-PAGE analysis of induction pattern of CspE and its mutants. Cells with pINIII vector alone, lane 1; pINIIIcspE, lane 2; pINIIIcspE-N7K-H32R, lane 3; pINIIIcspE-N7K, lane 4; and pINIIIcspE-H32R, lane 5. The band corresponding to overexpressed CspE is indicated.

Absence of Antitermination Is Not Due to the Destabilization of CspE by Mutations-- The apparent lack of antitermination by the double mutant CspE and by the H32R mutant may be due to destabilization of the CspE structure. Biophysical methods were used to ascertain that this is not the case. As seen from Fig. 2A, the secondary structure CD spectra of N7K and H32R mutant proteins were similar to that of wild-type CspE, with a minimum around 217 nm, which is characteristic of an unstacked -strand structure. The double mutant spectra varied a little, however, this difference in the CD spectra is marginal. The tertiary structure profile shown in Fig. 2B indicates that the double mutant and the H32R mutant are not significantly different in their tertiary packing than the wild-type CspE; however, mutant N7K showed more differences compared with the wild-type CspE. Finally, analysis of the melting patterns (Fig. 2C) showed that the H32R mutant was less stable (T_m = 51 °C) than wild-type CspE (T_m = 62 °C), whereas mutant N7K was more stable (T_m = 68 °C) than the latter. Interestingly, the double mutant showed stability (T_m = 62 °C) similar to that of the wild-type CspE. The effect of the mutations on the transcription antitermination seen is not a manifestation of the differences in the stability of these proteins, because (i) in the present communication, the effect of CspE mutations on antitermination of transcription was checked at 25-37 °C, wherein the stability of mutant proteins was not significantly different from that of the wild-type CspE, and (ii) the double mutant with stability similar to that of the wild-type CspE did not show transcription antitermination activity.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Circular dichroism (CD) and Tm of E. coli CspE and its mutants. A, secondary structure CD was measured from 140 to 260 nm. B, tertiary structure CD was measured from 260 to 320 nm. C, thermal folding of CspE and its mutants was monitored using changes in negative ellipticity at 222 nm. Wild-type CspE, open circles; CspE-N7K-H32R, closed circles; CspE-N7K, open triangles; CspE-H32R, closed triangles.

The H32R Substitution Results in Loss of Transcription Antitermination in Vitro-- Next we attempted to further characterize the effect of H32R mutation on transcription antitermination in vitro by using purified proteins. Stalled E. coli RNA polymerase elongation complexes were prepared on DNA template containing the T7 A1 promoter followed by a -independent terminator tR2 (26). Transcription was resumed by the addition of NTPs in the presence or in the absence of CspE proteins. As reported previously and as seen from Fig. 3, the wild-type CspE decreased transcription termination at tR2. The N7K mutant protein also decreased transcription termination, but the double mutant and the H32R mutant had no effect. The mean readthrough efficiency values (RE), defined as the fraction of the run-off transcripts of the total transcripts produced, were calculated for three independent experiments and are presented in Fig. 3. As can be seen, RE was less than 30% in the control reaction in the absence of CspE, in the presence of the double mutant and the H32R mutant. Addition of the wild-type CspE or the N7K mutant protein resulted in increase in the RE values to 55 and 48%, respectively. These results are consistent with the in vivo data shown above and suggest that single amino acid substitution of Asn at CspE position 7 with Lys does not affect the ability of CspE to cause transcription antitermination. In contrast, the substitution of His at CspE position 32 with Arg abolishes this activity.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   CspE mutations affect transcription antitermination in vitro. The in vitro transcription assays were carried out as described under "Experimental Procedures." DNA template containing the T7A1 promoter fused to the tR2 terminator was used, and CspE or its mutants were included in the reaction mixtures. The products were analyzed by urea-PAGE (7 M urea/10% polyacrylamide). RO and tR2 indicate the runoff and the tR2-terminated transcripts, respectively. Lower panel, results of quantification of the gel shown at the top panel. The readthrough efficiency was calculated as described under "Results" and "Discussion."

Transcription Antitermination Defect of CspE Mutants Is Due to the Reduced RNA Melting Activity-- In E. coli, an intrinsic transcription terminator signal is encoded in RNA and consists of two essential elements: a stem loop structure followed by a stretch of U residues. To act as a transcription antiterminator, CspE presumably must bind the nascent RNA. Thus, the defect in CspE-induced antitermination can be the trivial consequence of the absence of RNA binding. CspE can act as an "RNA chaperone" destabilizing RNA secondary structures and increasing RNA susceptibility to RNase attack (17). Therefore, the defect in CspE-induced antitermination can also be due to defects in RNA melting by mutant CspE. To select between these two possibilities, we tested whether CspE mutants were impaired in their (i) RNA-binding activity or (ii) RNA-melting activity.

We used an 88-nucleotide-long radioactively labeled RNA to check the RNA binding of CspE and its mutants by filter binding. The RNA fragment was previously selected as a preferred substrate of CspE binding by SELEX (18). The apparent K_d value of the binding reaction was defined as the concentration of protein at which half of maximum binding was obtained as described by Kajita et al. (27). As seen from Fig. 4, the K_d value for CspE was 0.3 × 10⁶ M. These values are consistent with the previous report (18). The calculated K_d values for the double mutant, and N7K and H32R mutants were 0.35, 0.06, and 0.09 × 10⁶ M, respectively. Thus, the double mutant bound the RNA substrate with approximately the same efficiency as the wild-type CspE. On the other hand, both the N7K and the H32R mutants showed significantly higher RNA binding than the wild-type CspE. It is not clear at this point how a combination of the two single mutations that increase CspE binding to RNA results in lower, "wild-type" binding efficiency. Be as it may, the results suggest that the inability of CspE carrying the H32R substitution to elicit transcription antitermination is not due to decreased RNA binding activity.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   RNA binding of CspE and its mutants. Filter binding assays (see "Experimental Procedures") were preformed using variable amounts of each protein and constant amount (50 fmol) of the 32P-labeled RNA probe. Wild-type CspE, open squares; CspE-N7K-H32R, open triangles; CspE-N7K, closed squares; CspE-H32R, open circles. The values shown are the mean values of three independent binding experiments.

To determine the effects of CspE mutations on nucleic acid melting we used molecular beacon system designed by Tyagi and Kramer (28). The molecular beacon is a single-stranded nucleic acid molecule that possesses a stem and a loop structure. A fluorescent moiety is attached to the end of one arm, and a nonfluorescent quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, resulting in efficient quenching of the fluorophore fluorescence. When a protein "opens up" the hairpin loop structure, the arms of the beacon move apart causing the fluorophore and the quencher to move away from each other as well. Because the fluorophore is no longer in close proximity to the quencher, its fluorescence will increase. Purified CspE was added to the beacon, and the fluorescence was monitored as described under "Experimental Procedures." As seen from Fig. 5A, the addition of the wild-type CspE resulted in the stably increased beacon fluorescence. Next, the experiment was repeated, but trypsin was added to the reaction mixture after the maximum fluorescence was achieved. This resulted in a slow decrease in the fluorescence as the tryptic degradation of CspE occurred, because the two arms of the beacon came back together bringing back the quencher in close proximity of the fluorophore again. A control experiment showed that CspE alone did not have any fluorescence. We conclude that (i) increased fluorescence of the beacon is due to nucleic acid melting caused by CspE, and (ii) molecular beacon system is suitable to study in vitro nucleic acid-melting activity of CspE and other CspA homologues.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Nucleic acid-melting activity of CspE and its mutants. A, the excitation and emission wavelengths used were 555 and 575 nm, respectively. The fluorescence of an 82-nucleotide long molecular beacon was monitored as wild-type CspE was added, as described under "Experimental Procedures." The effect of CspE removal was tested by adding trypsin in 50 times excess to the reaction mixtures. Time points at which the respective proteins were added are shown with arrows. Fluorescence of CspE itself was checked by adding CspE in the buffer without the molecular beacon. CspE, open circles; CspE and trypsin added, open squares; CspE added in buffer without molecular beacon, closed circles. B, the fluorescence of the molecular beacon was monitored as the CspE or its mutants were added. The relative fluorescence obtained with each of the proteins is shown (100% equals fluorescence obtained with the wild-type CspE).

The molecular beacon experiment was repeated with CspE mutants. For each of the mutant proteins, the maximum fluorescence value was obtained at the 0.25 × 10³-s time point, as was the case for the wild-type CspE (data not shown, and Fig. 5A). In Fig. 5B, the relative fluorescence obtained with each protein at 0.25 × 10³-s time is shown (the fluorescence value obtained with the wild-type CspE, was assumed as 100%). As can be seen, the N7K mutant showed 78% fluorescence as compared with CspE, whereas the double mutant and the H32R mutant showed 20% of the wild-type activity. Increasing amounts of any of the proteins did not further increase the fluorescence obtained, and none of the mutant proteins showed fluorescence by themselves (data not shown). We conclude that the H32R mutation results in reduced nucleic acid-melting activity of CspE.

Previously the RNA-secondary structure-destabilizing activity of CspE was shown by using an RNA corresponding to the 5'-untranslated repeat region of cspA (20). Analysis of CspE mutants using this method indicated that the wild-type and N7K mutant CspE destabilized the secondary structures in the RNA, whereas the double mutant and the H32R mutant did not (data not shown). The present results thus indicate that the ability of CspE to cause transcription antitermination and to destabilize nucleic acid secondary structure is correlated, providing the best evidence so far that Csp proteins target the stem-loop structure of the nascent RNA when functioning as transcription antiterminators.

Physiological Significance of the Functional Aberrations of CspE-- CspE may be involved in different cellular functions, some of them being manifestations of its RNA-binding activity alone and others requiring additional activities such as the RNA-melting activity. We used CspE mutants to check whether nucleic acid-melting function is relevant for two of the in vivo functions of CspE.

It has been shown that CspC and CspE are involved in the regulation of expression of E. coli rpoS, a gene coding for the stationary phase sigma factor, ^S. This regulation is mainly at post-transcriptional level, and mRNA stabilization is one of the important aspects of this regulation. It is believed that mRNA stabilization is due to Csp binding to the rpoS mRNA (16). Western blot analysis using monoclonal anti-^S antibodies showed that overexpression of the wild-type CspE resulted in a ~4-fold up-regulation of rpoS, as expected (Fig. 6, see also Ref. 16). Overexpression of any of the three CspE mutants resulted in a similar up-regulation of rpoS (Fig. 6). Therefore, we conclude that nucleic acid melting and/or transcription antitermination functions of CspE are not involved in the rpoS up-regulation.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   RpoS levels in cells overexpressing CspE and its mutants. Results of the Western blot analysis of RpoS levels in the JM83 E. coli cells transformed with pINIII vector alone, and with pINIII-based plasmids overproducing the wild-type CspE, CspE-N7K-H32R, CspE-N7K, CspE-H32R are presented. An equal number of cells was applied to each lane. Blots were probed with anti-RpoS monoclonal antibodies.

Next we examined if nucleic acid melting and/or transcription antitermination functions of CspE have any relevance to its ability to complement the cold-sensitive phenotype of the quadruple deletion strain of E. coli. E. coli CspA family consists of nine homologous members. The functions of family members appear to be partially redundant. A quadruple deletion strain cspAcspBcspGcspE exhibits cold sensitivity at 15 °C, which is complemented by overproduction of any one of CspA homologues except CspD. We introduced pINIII-based wild-type CspE, mutant CspE-overproducing plasmids, or the pINIII vector alone in the quadruple deletion strain and growth at 15 and 37 °C was examined on LB plates with and without 0.1 mM IPTG. The low levels of IPTG were used, because higher concentrations of the inducer were found to be toxic to cspE plasmid-bearing cells.

As seen from Fig. 7, all clones grew at 37 °C, except that the growth of cells carrying the pINIIIcspE-N7K plasmid was inhibited in the presence of IPTG, suggesting that overproduction of CspE-N7K is toxic for this particular deletion strain. As reported previously, the pINIII vector allows leaky expression of a cloned gene even in the absence of inducer and similar inhibition of colony formation in the presence of IPTG was seen with CspB-, CspC-, and CspH-overproducing plasmids (14). Toxicity associated with the overproduction of CspE-N7K was not seen in the E. coli strains such as the wild-type JM83 strain or the RL211 strain. Furthermore, overproduction of the double-mutant CspE had no adverse effect on cell growth at 37 °C (Fig. 7), as if the H32R substitution modulated the toxic effect of the N7K substitution. It is possible that both the toxicity of induced cspE-N7K and the ability to complement the Cs phenotype when uninduced are simply due to higher levels of cspE-N7K expression. Indeed, quantitative analysis of the wild-type and mutant CspE overproduction showed that CspE-N7K was consistently overproduced to higher levels than other CspE proteins (~1.5-fold higher, data not shown).

View larger version (58K): [in this window] [in a new window]   Fig. 7.   Effect of mutations within CspE on its ability to complement the cold-sensitive phenotype of the quadruple deletion strain of cspAcspBcspGcspE. The indicated pINIII-based cspE expression plasmids and the pINIII vector control were transformed in the quadruple csp deletion E. coli, and cells were streaked on LB plates with and without 0.1 mM IPTG as indicated by the diagram on the left. The plates were incubated at 15 and 37 °C. The results of the overnight growth at 37 °C and 3-day growth at 15 °C are presented.

Consistent with the previous report (14), plasmid overproducing wild-type cspE complemented the low temperature growth defect of the quadruple deletion strain, only in the presence of IPTG, whereas cells with pINIII vector alone did not form colonies at 15 °C either in the presence, or in the absence of IPTG. Similarly, plasmids overexpressing the double mutant and the H32R mutant did not complement the growth defect of the quadruple deletion strain. In contrast, cells carrying pINIIIcspE-N7K exhibited growth at 15 °C but only in the absence of IPTG, consistent with the result seen at 37 °C. This suggests that nucleic acid-melting/transcription antitermination activities of CspE are critical for its function for cold acclimation of the cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite the extensive research on CspA and its homologues, it is not known exactly how these proteins help the cold acclimation of cells. At low temperatures, the secondary structures of RNA stabilize, which should slow down transcription elongation and ribosomal movement on RNA. Csps, acting like "RNA chaperones" could destabilize the secondary structures in RNA and thus facilitate transcription and translation (17). In this paper we show that a CspE mutant selected for its inability to antiterminate transcription at high temperature is unable to complement cold sensitivity of quadruple csp deletion. In vitro, the mutant CspE protein binds RNA normally but is defective in destabilizing nucleic acid secondary structures. Thus, the RNA-chaperoning activity of CspE is essential for transcription antitermination function of the protein. Previously, we had shown that, in addition to reducing the efficiency of transcription termination on -independent terminators, Csps reduce hairpin-induced transcription pausing. Taken together, the data demonstrate that CspE affects transcription termination by preventing the formation of the nascent RNA secondary structures, most likely the formation of the stem-loop structure at the terminator. Thus, the mechanism of Csp-induced antitermination appears to be similar to the recently deduced mechanism of transcription antitermination by  N protein (29).

Previously we showed that (i) Csp proteins can act as transcription antiterminators in vivo and in vitro; (ii) at cold-shock, expression of promoter distal genes of the metY-rpsO operon, nusA, infB, rbfA, and pnp, is increased; and (iii) Csp overproduction causes similar increase in promoter distal metY-rpsO operon gene expression even at high temperature (20). This result provided the first evidence that transcription antitermination function of cold-shock-induced Csps is relevant, because the products of nusA, infB, rbfA, and pnp, which are NusA, IF2, RbfA, and PNP, respectively, are known to be induced at cold-shock and presumably enable the cells to adapt to low temperature. Our current results demonstrate that RNA-melting function of Csps is necessary for cellular adaptation to cold and are consistent with the idea that transcription antitermination function of Csps is linked to their cold-shock function. On the other hand, another function of CspE, its ability to up-regulate the rpoS gene expression, is unaffected by termination-altering mutations and is probably dependent only on its RNA-binding activity.

The three-dimensional structure of CspA from E. coli has been resolved by x-ray crystallography and NMR analysis (30-32). The structure consists of five antiparallel -strands, 1 through 5, forming a -barrel structure with two -sheets. The two evolutionarily conserved RNA-binding motifs of CspA, RNP1 and RNP2, are located on the 2 and 3 strands. RNP1 Phe¹⁸ and Phe²⁰ and RNP2 Phe³¹ and His³³ form a compact surface-exposed site on the CspA three-dimensional structure. These residues are presumably involved in intercalation between the bases of a nucleic acid target. Mutational analysis of E. coli CspA showed that single substitutions of Phe residues at positions 18, 20, and 31 by either Leu or Ser residues decreased DNA binding (33). Similarly, in the case of CspB from Bacillus subtilis, which is a homologue of E. coli CspA, substitution of Phe residues at positions 15, 17, and 27 by Ala and substitution of His at position 29 by Gln also abolished nucleic acid binding (34). Here, we show that substitution of E. coli CspE His³², which corresponds to E. coli CspA His³³ and B. subtilis CspB His²⁹, for Arg does not affect the RNA-binding activity, probably because Arg is capable of favorable electrostatic interaction with nucleic acids (34). The loss of the RNA-melting activity in H32R CspE mutant may be due to the difference in the side chains of His and Arg, because the imidazole ring of His is capable of intercalation between nucleic acid bases, whereas the guanidine group of Arg is not. According to this view then, in the wild-type CspE, His³² could participate in the initiation of nucleic acid secondary structure melting, which is further propagated by subsequent intercalation of Phe¹⁸, Phe²⁰, and Phe³¹. Our ongoing site-specific mutational analysis of cspE and integrated biophysical, biochemical, and physiological analyses of CspE mutants should clarify this issue.

	ACKNOWLEDGEMENTS

We thank Dr. Sanjay Tyagi for the gift of molecular beacons and also for useful suggestions. We also thank Dr. Ujwal Shinde for the CD and T_m measurements. We are grateful to Dr. S. Nechaev for providing the materials and for advice with the in vitro transcription assays.

	FOOTNOTES

^* This work was supported by National Institutes of Health (NIH) Grant GM19043 (to M. I.) and by the Burroughs Welcome Fund Career Development Award and NIH Grant GM59295 (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence may be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-4115; Fax: 732-235-4559; E-mail: inouye@umdnj.edu.

To whom correspondence may be addressed: Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik@waksman.rutgers.edu.

Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M111496200

	ABBREVIATIONS

The abbreviations used are: Csp, cold-shock protein; LB, Luria-Bertani broth; IPTG, isopropyl -D-thiogalactopyranoside; CD, circular dichroism; EC²⁰, elongation complex stalled at position +20; SELEX, systematic evolution of ligands by exponential enrichment; RE, readthrough efficiency.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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