Mol. Cells, Vol. 19, No. 2, pp. 239-245

Molecules and Cells KSMCB 2005

Effects of FIS Protein on rnpB Transcription in Escherichia coli Hyun-Sook Choi, Kwang-sun Kim, Jeong Won Park, Young Hwan Jung, and Younghoon Lee* Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea. (Received November 9, 2004; Accepted December 16, 2004)

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs. There are multiple putative FIS binding sites upstream of the rnpB promoter. We tested whether FIS binds to these sites, and if so, how it affects rnpB transcription. In vitro binding assays revealed specific binding of FIS to multiple sites in the rnpB promoter region. Interestingly, FIS bound not only to the upstream region of the promoter, but also to the region from +4 to +18. FIS activated rnpB transcription in vitro, but the level of activation was much lower than that of the rrnB promoter for rRNA. We also examined the effects of FIS on rnpB transcription in vivo using isogenic fis+ and fis− strains. rnpB transcription was higher in the fis− than the fis+ cells during the transitions from lag to exponential phase, and from exponential to stationary phase. Keywords: E. coli; FIS; M1 RNA; Protein-DNA Interaction; rnpB; Transcription.

Introduction Factor for inversion stimulation (FIS) is a nucleoidassociated and site-specific DNA-binding protein that also functions as a regulator of gene expression in Escherichia coli (Betermier et al., 1994; Dorman and Deighan, 2003; Finkel and Johnson, 1992; Muskhelishvili and Travers, 2003). The rRNA genes have multiple FISbinding sites, one of which is present in an upstream acti* To whom correspondence should be addressed. Tel: 82-42-869-2832; Fax: 82-42-869-2810 E-mail: [email protected]

vator sequence (UAS) (Hirvonen et al., 2001). The UAS has a primary site located at position -71 with respect to the transcription start site. The UAS-bound FIS contacts the carboxyl-terminal domain of the α-subunit of RNA polymerase and activates transcription (Hirvonen et al., 2001; Ross et al., 1990; Zhi et al., 2003). FIS also affects transcription from tyrT by enhancing wrapping of the upstream DNA around RNA polymerase and helping it to overcome the resistance to promoter melting due to the G+C-rich discriminator sequence between -10 and the transcription start site (Muskhelishvili et al., 1997; Pemberton et al., 2002). FIS can also act as a repressor. It negatively regulates the promoter responsible for transcription of its own gene (Walker et al., 1999) and the gyrA and gyrB genes encoding DNA gyrase (Schneider et al., 1999). Since FIS activates one of the promoters of topA, coding for topoisomerase I (Weinstein-Fischer et al., 2000), its effects on transcription are related to the topology of chromosomal DNA. FIS is abundant under conditions in which stationary cells are committed to start active growth. The maximum level of FIS of 50,000−100,000 copies per cell (Ball et al., 1992), occurs in the early exponential growth phase. Fis− cells have reduced growth rates and morphological abnormalities (Filutowicz et al., 1992; Spaeny-Dekking et al., 1995). FIS can bend DNA through nonspecific binding and directly affect the topology of the bacterial DNA (Betermier et al., 1994; Muskhelishvili and Travers, 2003; Pan et al., 1996; Schneider et al., 1999). It also accelerates the accumulation of moderately supercoiled plasmids in stationary phase (Schneider et al., 1997). Therefore, it appears to participate in the regulation of transcription by modulating chromosomal dynamics over the bacterial growth cycle. M1 RNA, the catalytic subunit of E. coli RNase P (Guerrier-Takada et al., 1983), is also a stable RNA, and can be regarded broadly as a component of the translational apparatus like tRNA or rRNA because RNase P is a

240

Effects of FIS on rnpB Transcription

processing enzyme involved in the maturation of tRNAs (Robertson et al., 1972). Therefore, it is reasonable to suppose that M1 RNA biosynthesis is regulated in a manner similar to biosynthesis of rRNA or tRNA. Indeed, transcription from the rnpB gene encoding M1 RNA is inhibited during the stringent response, like rRNA and tRNA genes (Jung and Lee, 1997; Lee et al., 1991). The rnpB promoter has a GC-rich discriminator sequence between the −10 element and the transcription start site that is responsible for the stringent control of stable RNA synthesis (Jung and Lee, 1997). rnpB also has several putative FIS-binding sites upstream of the rnpB promoter, but it was not known whether FIS bound to these sites, and if so, how this binding affected rnpB transcription. In this study, we show that FIS binds to several sites in the rnpB promoter in vitro. Interestingly, it also binds to the region from +4 to +18. However, transcriptional activation of the rnpB promoter by FIS was much lower than that of the rrnB promoter in vitro. In vivo, the effects of FIS on rnpB transcription were complex. RnpB transcription was slightly higher in fis− than in fis+ cells in the transition from the lag phase to exponential growth. We suggest that FIS plays a role in the tight, growth-dependent regulation of rnpB transcription by causing global changes of local chromosomal structure at the rnpB locus.

Materials and Methods Bacterial strains and plasmids The E. coli K-12 strain JM109 was used for constructing plasmids. Strain MC1000 (λrnpB-lacZ1), which carries the lacZ gene fused to the rnpB promoter (Jeon et al., 1993), was used for analyzing rnpB transcription. We constructed a fis- strain (fis::km) in the MC1000 (λrnpB-lacZ1) background by inserting the kanamycin resistance gene (kmR), as described previously (Yu et al., 2000). Briefly, a kanamycinresistance linear cassette was synthesized by PCR using primers 1 (5′-AAA TTC TGA CGT ACT GAC CGT TTC TAC CGT TAA CTC TCA GGA TCA GGT AAT ATG GAC AGC AAG CGA AC-3′) and 2 (5′-ACG GTT GAT GCC CAT CAT CAG CGC AGC ACG GGT CTG GTT ACC ACG GGT GTT CAG AAG AAC TCG TCA AGA AG-3′) from Tn5. The linear cassette was introduced into strain DY330 [W3110 ∆lacU169 gal490 λcl857 ∆(cro-bioA)] (Yu et al., 2000) to generate DY330-fis::km, and the fis::km construct was transferred to strain MC1000(λrnpB-lacZ1) by P1 transduction to generate MC1000(λrnpB-lacZ1)-fis::km. Plasmid pLMd23 is a derivative of pGEM, which contains the rnpB sequence from −270 to +1286, together with an internal deletion of the sequence between +57 and +330 in the M1 RNA structural gene (Kim et al., 1996). We constructed pLM-rrnB by replacing the −270 to +56 region of the rnpB promoter with the −272 to +78 region of the rrnB promoter. Expression and purification of FIS We constructed pET-fis,

an expression plasmid for FIS, by inserting the FIS-encoding DNA fragment carrying an NdeI site before the start codon and a BamHI site after the stop codon into the NdeI/BamHI sites of pET21a (Novagen). E. coli BL21(DE3)pLysS cells carrying pET-fis were grown in LB containing ampicillin (100 µg/ml) at 37°C to A600 of about 0.7, and FIS expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 1 h. The cells were harvested (about 2 g in wet weight), resuspended in 25 ml lysis buffer [50 mM Tris-HCl, pH 8.0, 10% sucrose, 2 mM DTT, 15 mM EDTA, 200 µg lysozyme per ml, 100 µl protease inhibitor cocktail (Sigma) per ml, 300 mM NH4SO4], and sonicated on ice. The supernatant was recovered by centrifugation, dialyzed against 0.3 M NaCl column buffer (0.3 M NaCl, 20 mM Tris-HCl, pH. 8.0, 0.1 mM EDTA), and subjected to SPSepharose (Amersharm) chromatography. Proteins were eluted with column buffer with a linear gradient of 0.3–1 M NaCl. Fractions containing FIS were dialyzed against a 0.1 M NaCl column buffer and further purified by gel filtration using a Hiload superdex 75 column (Amersham) according to the manufacturer’s instructions. The eluted protein was dialyzed against 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA, and glycerol was added to samples to a final concentration of 50% for storage at –20°C. Gel-mobility shift assay Gel-mobility shift assays were carried out as previously described (Kwon et al., 2004), with minor modifications. Briefly, a −200 to +40 fragment of the rnpB promoter was synthesized by PCR amplification using primers rnpB-f (5′-ACCGATGATGTTGGC) and rnpB-r (5′-GAAGAGGACGACGAC) and labeled with 32P. The primers were 5′ labeled with polynucleotide kinase and [γ-32P] ATP (3000 Ci/mmole). 0.1 ng of labeled PCR products were preincubated for 10 min at 37°C in binding buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 mg/ml BSA, 10 µg/ml salmon sperm DNA, and 5% glycerol). FIS protein was serially diluted to the desired concentration in the same buffer, and the binding assay was initiated by adding FIS protein to the DNA-containing solution in a final volume of 20 µl. As controls, an excess of unlabeled rnpB promoter fragments or salmon sperm DNA were added to binding reactions. After 20 min at 37°C, 10 µl of each reaction mixture was loaded onto a 5% nondenaturing polyacrylamide gel (acrylamide: bisacrylamide = 29:1). After electrophoresis, the gel was dried and analyzed with a Fuji Bio-Imaging Analysis System 1500 (BAS-1500). DNase I footprinting The same rnpB promoter-containing fragment that was used for gel-mobility shift assays was also used for DNase I footprinting. The DNA fragment was PCRamplified with 5′ labeled rnpB-f and unlabeled rnpB-r primers or vice versa and incubated with FIS protein at 37°C in 25 µl of binding buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA). After a 20-min incubation, 100 ng of DNase I was added, and the reaction mixture was incubated for 30 s. The reaction was stopped by phenol extraction, and the DNA was precipitated with ethanol, electro-

Hyun-Sook Choi et al.

241

phoresed on an 8% polyacrylamide sequencing gel and analyzed as above. In vitro transcription Two ng pLMd23 or pLM-rrnB plasmid DNA as template was pre-incubated for 10 min at 37°C with varying concentrations of FIS in 15 µl of reaction buffer (70 mM KCl, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA) and NTP mix (5 mM ATP, 0.5 mM UTP, 0.1 mM GTP, 0.1 mM CTP, 1 µCi [α-32P]CTP). Transcription was initiated by adding 0.06 units of RNA polymerase (Amersham) in 5 µl of reaction buffer. After a 20-min incubation, the reaction was terminated by adding 5 µl of stop solution (7 M urea, 25 mM EDTA, 2.5% SDS, 5× TBE, 0.1% BPB, 0.05% XC, 10% glycerol), and products were analyzed on 5% polyacrylamide sequencing gels and analyzed as above. +

−

Primer extension Isogenic fis and fis cells were grown overnight in LB containing 50 µg/ml ampicillin at 37°C, diluted 1:100 in the same medium and grown with shaking at 37°C. Aliquots of the cultures were taken at intervals to determine growth and isolate total cellular RNA. The latter was extracted by the hot-phenol extraction method (Kim et al., 1996). rnpBCAT-lacZ fusion transcripts were measured by primer extension with primer R-cat (5′-TTGTCCTACTCAAGCTTGGCTGCAGGTCGA-3′), which is complementary to the CAT-coding sequence. For primer-extension, we labeled the primer with T4 polynucleotide kinase and [γ-32P]ATP at the 5′ end. To carry out the primer-annealing reaction, we mixed 0.6 pmole of the labeled primer with about 30 µg total RNA in 10 µl AMV RT buffer (Promega). The primer/RNA mixture was incubated at 65°C and allowed to cool to 25°C. After annealing, we added 3 µl of 5× AMV RT buffer (Promega), 1.5 µl of 20 mM dNTP mix, 1 µl of RNasin (40 U, Promega), 2 µl of AMV RT (40 U, Promega), and 7.5 µl of RNase free water. The reaction mixture was incubated at 42°C for 1 h and the extension products were electrophoresed on a 5% polyacrylamide sequencing gel and analyzed as above.

Results and Discussion Interaction of FIS with the rnpB promoter Transcription of rnpB shares many features with transcription of rrnB. Both processes, which produce stable RNAs, are growth rate- and growth phase-dependent (Condon et al., 1995; Jeon et al., 1993; Park et al., 1996), and repressed by the stringent response (Cashel et al., 1996; Jung and Lee, 1997). Both promoters have GC-rich discriminator regions between the -10 region and the transcription start site. Since FIS activates rrnB transcription (Bokal et al., 1997; Gosink et al., 1996), we tested whether it also affected rnpB transcription. A gel mobility shift assay (Fig. 1) with a labeled −200 to +50 DNA fragment of rnpB yielded multiple bands, suggesting the presence of multiple binding sites. These bands were competed by unla-

Fig. 1. Interaction between FIS and the rnpB promoter. A 32Plabeled rnpB promoter-containing DNA fragment of 0.08 nM (about 2.75 ng) was incubated with increasing concentrations of FIS. The presence of unlabeled rnpB promoter-containing DNA fragments and salmon sperm DNA as specific and nonspecific competitors, respectively, is indicated.

beled rnpB promoter but not by salmon sperm DNA. Next, we sought to identify the FIS binding sites in the rnpB promoter by DNase I footprinting. FIS binding sites were identified by comparing the DNase I footprinting patterns with the consensus sequence for FIS binding (Finkel and Johnson, 1992). Four regions homologous to the consensus sequence were defined as FIS binding sites (Fig. 2). They were located in regions −163 to −149, −125 to −111, −57 to −43, and +4 to +17. However, we do not exclude the possibility that there are additional FIS binding sites, because 6 shifted bands were detected in the gel mobility shift assay. The distribution of the FIS-binding sites differed from that in the rrnB promoter (Fig. 3). First, there was no FIS-binding site in the −70 region whereas this is the region of the rrnB promoter where bound FIS interacts with α-CTD of RNA polymerase to stabilize the initiation complex (Bokal et al., 1997; Gosink et al., 1996). Second, there is a FIS-binding site from +4 to +17, downstream of the transcription start site, while only upstream FIS-binding sites were detected in the rrnB promoter. These differences suggest that binding of FIS to the two promoters may affect transcription in a different manner. Effects of FIS on rnpB transcription in vitro In order to examine how binding of FIS to the rnpB promoter affects rnpB transcription, we examined in vitro transcription with E. coli RNA polymerase in the presence of FIS (Fig. 4). pLMd23 plasmid DNA, which was used as template, generates a truncated M1 RNA transcript of 147 nucleotides. We assessed the efficiency of transcription of the rnpB promoter as the ratio of the amount of M1 RNA

242

A

Effects of FIS on rnpB Transcription

B

Fig. 2. DNase I probing of the interaction between FIS and the rnpB promoter. 32P end-labeled rnpB promoter-containing fragments between positions −270 and +50 were prepared by PCR amplification. One of the primers was labeled with 32P at the 5′ end and the other was unlabeled. The 5′-labeled DNA fragments at positions −200 (A) and +50 (B) were used for DNase I footprinting by incubating them with increasing amounts of FIS at 37°C for 20 min, followed by partial DNase I digestion. The treated DNA was electrophoresed on a 5% polyacrylamide sequencing gel and analyzed with a BAS 1500 (Fuji). Lanes 1−7 correspond to 0, 38, 76, 150, 380, 760, and 1910 nM FIS. The FIS binding sites are indicated.

transcript to that of RNA I, which is also transcribed from the plasmid. The rrnB promoter, which generates an rrnB transcript of 150 nt, was used as a control, and we examined the effect of varying the salt concentration, because it is reported that rrnB transcription is not activated in low salt (Ross et al., 1990). FIS activated rnpB transcription most effectively at moderate salt concentrations (70 mM KCl), as was the case for rrnB transcription. However, it was much less effective in activating rnpB transcription than rrnB transcription. Also, activation by FIS in the presence of 70 mM of KCl increased with increasing FIS to a maximum at 120 nM. At this FIS concentration, rnpB transcription was about 1.5-fold stimulated. This small degree of stimulation contrasts with a larger extent of activation (about 3-fold) of rrnB transcription. At higher FIS concentrations rnpB transcription was inhibited whereas rrnB transcription was sustained. This difference may be related to the differences in the FIS binding patterns between the two promoters. The presence of a FISbinding site downstream of the transcription start site in the rnpB promoter could reduce FIS activation and cause inhibition at high FIS concentrations. However, we cannot

Fig. 3. Comparison of FIS-binding sites in the rnpB and rrnB promoters. The FIS-binding sites are underlined. The −10, −35 regions and UP element are indicated by boxes, and the discriminator region is underlined. The consensus sequences for FIS binding [Gnn(c/t)(A/g)(a/t)(a/t)(T/A)(t/a)(t/a)(T/c)(g/a)nnC], where n is any nucleotide (Finkel and Johnson, 1992) are indicated by asterisks.

exclude the possibility that the marginal FIS effects on the rnpB transcription are due to other factors, such as impurities in the protein preparation. FIS effects on in vivo rnpB transcription We next determined the effect of FIS on rnpB transcription in vivo using a FIS-deficient mutant. To construct the mutant strain, we inserted the kanamycin-resistance gene (kmR) into the fis gene into an E. coli lysogen, MC1000 (λrnpBlacZ1) carrying lacZ fused to the rnpB promoter. The rnpBlacZ fusion also included a translation signal sequence corresponding to part of the N-terminal coding sequence of the chloramphenicol acetyltransferase (CAT) gene (Jeon et al., 1993). We prepared total RNAs from fis− and fis+ cells at different stages of growth and subjected them to primer-extension analysis using a primer complementary to the CAT-coding sequence (Fig. 5). We also performed primer extension analysis with different amounts of total RNA, in order to relate the amount of primer-extension product to the RNA content (data not shown), and used

Hyun-Sook Choi et al.

A

243

A

B B

Fig. 4. FIS effects on in vitro transcription. In vitro transcription was carried out with E. coli RNA polymerase using pLMd23 as template. The resulting RNA was analyzed on a 5% polyacrylamide gel. pLM-rrnB containing the rrnB promoter served as a control template. Transcription reactions were performed in a reaction buffer containing 70 mM KCl. Lanes 1−6 correspond to 0, 30, 50, 120, 240, and 480 nM of FIS (A), or various KCl concentrations with 80 nM FIS (B). In vitro transcripts are indicated by arrows. Relative transcription activity was calculated by normalizing the amount of transcripts to that of RNA I, and the effect of FIS is represented as the ratio of the relative transcription activity in the presence of FIS to that in the absence of FIS.

the intracellular content of rnpB-CAT-lacZ fusion mRNA to assess the transcription rate. Since FIS is known to affect rRNA synthesis (Newlands et al., 1992; Nilsson et al., 1992), cellular RNA content is FIS-dependent. Therefore, we did not normalize the RNA amounts to quantify the rnpB transcription rate. Instead, we compared extension products using total cellular RNA isolated from the same number of cells. Since the amounts of the extension products may not accurately reflect relative rnpB transcription rates in the fis+ and fis− strains, we also compared the relative rnpB transcription rates between the two strains throughout the growth cycle. In both strains, rnpB transcription was high in the transition from the lag phase to the exponential phase, decreased during the exponential phase, and almost stopped before the transition from the exponential to the stationary phase. These findings differ somewhat from the results for transcription from rnpB in a multicopy plasmid, which increases rapidly during exponential growth, reaching a maximum in the mid-

Fig. 5. Analysis of rnpB transcription in vivo. A. fis+ and fis− isogenic cells were grown in LB medium at 37°C and total cellular RNA extracts were prepared from cells at different growth phases. The A600 values of the cultures used for RNA preparation are indicated above each lane. 30 µg total cellular RNA was used for primer extension analysis to determine the amounts of rnpB-CAT-lacZ fusion transcript, and the extension products were analyzed on a 5% polyacrylamide sequencing gel. B. Growth curves and relative amounts of rnpB-CAT-lacZ transcripts. Open and filled symbols represented fis+ and fis− cells, respectively. Values are from three independent experiments.

exponential phase and decreasing sharply in the late exponential phase (Park et al., 1996). The fis− cells entered the exponential phase slightly later than did the fis+ cells, and the transition from the exponential to the stationary phase was more extended in the fis− cells. rnpB transcription was more strongly stimulated in the fis− cells in the transition from lag to exponential phase. We also observed the up-regulation in fis− cells in the transition from exponential to stationary phase, at an A600 of 3. The observation that growth-phase-dependent regulation of rnpB transcription in fis− cells differs from that in fis+ cells also suggests that FIS is involved in growth-phase-dependent regulation of rnpB transcription. It has been proposed that FIS modulates chromosomal dynamics during bacterial growth (Schneider et al., 1997; 1999). Therefore, the different pattern of growth-phase-dependent regulation in fis− cells may result from a lack of growth phase-dependent topological changes of chromosomal dynamics (Schneider et al., 1999). We also found that growth-phase-dependent regulation of rnpB transcription from the bacterial chromosome differs from that of rnpB from a multicopy plas-

244

Effects of FIS on rnpB Transcription

mid even in fis+ cells. This difference could be explained by the hypothesis that FIS affects rnpB transcription via a local change of chromosomal topology at the rnpB locus during bacterial growth.

Acknowledgment This work was supported by a Korea Research Foundation Grant (KRF-2002- 015-CP0238).

References Ball, C. A., Osuna, R., Ferguson, K. C., and Johnson, R. C. (1992) Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. J. Bacteriol. 174, 8043−8056. Betermier, M., Galas, D. J., and Chandler, M. (1994) Interaction of Fis protein with DNA: bending and specificity of binding. Biochimie 76, 958−967. Bokal, A. J., Ross, W., Gaal, T., Johnson, R. C., and Gourse, R. L. (1997) Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter. EMBO. J. 16, 154−162. Cashel, M., Gentry, D. R., Hernandez, J. V., and Vinella, D. (1996) The stringent response in Escherichia coli and Salmonella; in Cellular and Molecular Biology, Neidhardt, F. C. (ed.), Vol. 1, pp. 23−54, Am. Soc. Microbiol., Washington, DC. Condon, C., Squires, C., and Squires, C. L. (1995) Control of rRNA transcription in Escherichia coli. Microbiol. Rev. 59, 623−645. Dorman, C. J. and Deighan, P. (2003) Regulation of gene expression by histone-like proteins in bacteria. Curr. Opin. Genet. Dev. 13, 179−184. Filutowicz, M., Ross, W., Wild, J., and Gourse, R. L. (1992) Involvement of Fis protein in replication of the Escherichia coli chromosome. J. Bacteriol. 174, 398−407. Finkel, S. E. and Johnson, R. C. (1992) The Fis protein: it’s not just for DNA inversion anymore. Mol. Microbiol. 6, 3257− 3265. Gosink, K. K., Gaal, T., Bokal, A. J. 4th., and Gourse, R. L. (1996) A positive control mutant of the transcription activator protein FIS. J. Bacteriol. 178, 5182−5187. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N., and Altman, S. (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849−857. Hirvonen, C. A., Ross, W., Wozniak, C. E., Marasco, E., Anthony, J. R., et al. (2001) Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli. J. Bacteriol. 183, 6305− 6314. Jeon, E. S., Lee, H. R., Lee, S. G., Jung, Y. H., Cho, M., et al. (1993) Growth rate-dependent regulation of the rnpB gene expression in Escherichia coli. Korean J. Genet. 15, 11−16. Jung, Y. H. and Lee, Y. (1997) Escherichia coli rnpB promoter mutants altered in stringent response. Biochem. Biophys. Res. Commun. 230, 582−586. Kim, S., Kim, H., Park, I., and Lee, Y. (1996) Mutational analysis of RNA structures and sequences postulated to affect 3′ processing of M1 RNA, the RNA component of Escherichia

coli RNase P. J. Biol. Chem. 271, 19330−19337. Kwon, C., Kwon, K., Chung, I. K., Kim, S. Y., Cho, M. H., et al. (2004) Characterization of single stranded telomeric DNAbinding proteins in cultured soybean (Glycine max) cells. Mol. Cells 17, 503−508. Lee, S. J., Jung, Y. H., Park, C.-U., and Lee, Y. (1991) Stringent control of Escherichia coli rnpB gene transcription in vivo in multicopy plasmids. Mol. Cells 1, 415−420. Muskhelishvili, G. and Travers, A. (2003) Transcription factor as a topological homeostat. Front. Biosci. 8, d279−285. Muskhelishvili, G., Buckle, M., Heumann, H., Kahmann, R., and Travers, A. A. (1997) FIS activates sequential steps during transcription initiation at a stable RNA promoter. EMBO J. 16, 3655−3665. Newlands, J. T., Josaitis, C. A., Ross, W., and Gourse, R. L. (1992) Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent. Nucleic Acids Res. 20, 719−726. Nilsson, L., Verbeek, H., Vijgenboom, E., van Drunen, C., Vanet, A., et al. (1992) FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions. J. Bacteriol. 174, 921−929. Pan, C. Q., Finkel, S. E., Cramton, S. E., Feng, J. A., Sigman, D. S., et al. (1996) Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. J. Mol. Biol. 264, 675−695. Park, J. W., Jung, Y. H., Park, B. H., Jeoung, Y. H., and Lee, Y. (1996) Direct analysis of the transcription of Escherichia coli rnpB gene harbored in a multicopy plasmid during bacterial growth. J. Biochem. Mol. Biol. 29, 221−224. Pemberton, I. K., Muskhelishvili, G., Travers, A. A., and Buckle, M. (2002) FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter. J. Mol. Biol. 318, 651−663. Robertson, H. D., Altman, S., and Smith, J. D. (1972) Purification and properties of a specific Escherichia coli ribonuclease which cleaves a tyrosine transfer ribonucleic and precursor. J. Biol. Chem. 247, 5243−5251. Ross, W., Thompson, J. F., Newlands, J. T., and Gourse, R. L. (1990) E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 9, 3733−3742. Schneider, R., Travers, A., and Muskhelishvili, G. (1997) FIS modulates growth phase-dependent topological transitions of DNA in Escherichia coli. Mol. Microbiol. 26, 519−530. Schneider, R., Travers, A., Kutateladze, T., and Muskhelishvili, G. (1999) A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol. Microbiol. 34, 953−964. Spaeny-Dekking, L., Nilsson, L., von Euler, A., van de Putte, P., and Goosen, N. (1995) Effects of N-terminal deletions of the Escherichia coli protein Fis on growth rate, tRNA(2Ser) expression and cell morphology. Mol. Gen. Genet. 246, 259− 265. Walker, K. A., Atkins, C. L., and Osuna, R. (1999) Functional determinants of the Escherichia coli fis promoter: roles of -35, -10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation. J. Bacteriol. 181, 1269−1280. Weinstein-Fischer, D., Elgrably-Weiss, M., and Altuvia, S. (2000) Escherichia coli response to hydrogen peroxide: a

Hyun-Sook Choi et al. role for DNA supercoiling, topoisomerase I and Fis. Mol. Microbiol. 35, 1413−1420. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., et al. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci.

245

USA 97, 5978−5983. Zhi, H., Wang, X., Cabrera, J. E., Johnson, R. C., and Jin, D. J. (2003) Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation. J. Biol. Chem. 278, 47340−47349.