Vol. 50 No. 4/2003

941–945

QUARTERLY

Communication

Non-random distribution of GATC sequences in regions of pro- moters stimulated by the SeqA of Escherichia coli.

Barbara Strzelczyk1, Monika Słomińska-Wojewódzka1, Grzegorz Węgrzyn1,2 and Alicja Węgrzyn3½

1Department of , University of Gdañsk, Gdañsk, Poland; 2Institute of Oceanology, Polish Academy of Sciences, Gdynia, Poland; 3Laboratory of Molecular Biology (affiliated with the University of Gdañsk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Gdañsk, Poland Received: 31 October, 2003; revised: 28 November, 2003; accepted: 03 December, 2003

Key words: SeqA protein, transcription regulation, promoter sequence, GATC motif

The SeqA protein of Escherichia coli is not only the main negative regulator of DNA replication initiation but also a specific . It binds to hemi- methylated GATC sequences and, with somewhat different specificity, to fully meth- ylated GATC regions. Recently, a microarray analysis was reported, in which transcriptomes of wild-type and DseqA strains were compared. Although in the seqA mutant the levels of some transcripts were significantly decreased while certain tran- scripts were evidently more abundant relative to wild-type , no correlation between the presence of GATC motifs in promoter sequences and transcription activ- ity was found. However, here we show that when larger DNA fragments, encompass- ing positions from –250 to +250 relative to the transcription start site, are analyzed, some common features of GATC distribution near the promoters activated by SeqA can be demonstrated. Nevertheless, it seems that the GATC pattern is not the only determinant of SeqA-dependence of promoter activity.

In Escherichia coli, the product of the seqA mosome replication initiation (Lu et al., 1994; gene is the main negative regulator of chro- von Freiesleben et al., 1994; Slater et al.,

.This work was supported by the State Committee for Scientific Research (KBN, Poland, grant No. 3 P04A 029 22 to A.W.). G.W. acknowledges financial support from the Foundation for Polish Science (subsidy 14/2000). ½ Corresponding author: Dr. Alicja Węgrzyn, Laboratory of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland; tel.: (48 58) 346 3014; fax: (48 58) 301 0072; e-mail: [email protected] 942 B. Strzelczyk and others 2003

1995; Boye et al., 1996; Wold et al., 1998). The ther decreased or increased in the seqA mu- SeqA protein is involved in sequestration of tant, while levels of transcripts of most genes the origin of replication, oriC,in were similar (less than two-fold difference) in the hemimethylated state (Lu et al., 1994; the wild-type and DseqA strains. Slater et al., 1995). This regulatory process is Since SeqA binds GATC sequences, a corre- crucial for precise control of bacterial cell lation between the presence of such motifs in cycle. the promoter sequence and stimulation or re- SeqA binds specifically to hemimethylated pression of transcription by the seqA gene GATC sequences located on oligonucleotide product was analyzed. Lobner-Olsen et al. fragments but no specific binding to analo- (2003) did not find any common features of gous fully methylated or unmethylated DNA the promoters whose activities appeared to be was reported (Brendler & Austin, 1999; Kang significantly changed in the seqA mutant rela- et al., 1999). However, when longer DNA frag- tive to wild-type bacteria. Therefore, those au- ments were employed, SeqA did bind a fully thors excluded the possibility that SeqA acts methylated DNA fragment bearing oriC directly at promoter sequences, and sug- strongly and specifically (Slater et al., 1995). gested that the decreased or increased tran- Moreover, hemimethylated oriC and non-oriC scription of certain genes in the seqA mutant sequences were bound by SeqA with similar may result from changes in nucleoid organi- affinity (Slater et al., 1995). Investigation of zation. However, contrary to that conclusion, SeqA-binding sites on hemimethylated oriC it was demonstrated experimentally that revealed the same preferential binding to SeqA stimulates transcription from certain each side of the DnaA box R1, as was found promoters both in vivo and in vitro, even for fully methylated oriC (Skarstad et al., when relatively short DNA templates were 2000). Thus, this binding has the same speci- used in in vitro experiments (Słomińska et al., ficity to certain GATC sites irrespective of 2001; 2003a; 2003b). These results strongly whether they are fully or hemimethylated. Ac- suggested that the SeqA protein is directly in- cording to this statement, it was demon- volved in the regulation of activity of certain strated that SeqA interacts specifically with promoters. Interestingly, analysis of distribu- certain bacteriophage l DNA fragments con- tion of GATC motifs near bacteriophage l taining GATC sequences both in the fully promoters, as well as the results of studies on methylated and hemimethylated state SeqA–DNA interactions at these regions, (Słomińska et al., 2001). strongly suggest that SeqA-binding sites Perhaps surprisingly, recent studies indi- downstream of a promoter may be important cated that apart from being the main negative for the SeqA-mediated stimulation of pro- regulator of DNA replication initiation, SeqA moter activity (Słomińska et al., 2003a; is also a specific transcription factor. Namely, 2003b). These sites can be located as far as this protein specifically stimulates transcrip- 100–200 bp from the transcription start site tion from bacteriophage l pR, pI and paQ pro- (Fig. 1). Such regions were not analyzed by moters (pI and paQ require also the action of Lobner-Olsen et al. (2003). Therefore, here we the l-encoded CII protein) both in vivo and in analyzed the distribution of GATC motifs in vitro, whereas some other l promoters (like regions of promoters (from position –250 to pL and pE) appear to be SeqA-independent +250) of the E. coli genes activated and re- (Słomińska et al., 2001; 2003a; 2003b). Very pressed in the seqA mutant (reported by recently, effects of DseqA mutation on the ex- Lobner-Olsen et al., 2003), and in randomly pression of all E. coli genes were studied us- selected genes whose transcription was simi- ing microarray analysis (Lobner-Olsen et al., lar in the wild-type and DseqA strains. 2003). Transcription of certain genes was ei- Vol. 50 SeqA-mediated transcription regulation 943

to find any patterns of the GATC motifs that might by obligatory for SeqA-mediated regu- lation of promoter activity, the distribution of these sequences near promoters that are de- pressed in seqA mutants (thus, potentially ac- tivated by SeqA) seems to be non-random (Fig. 2A). In a relatively large fraction of these promoters (9 out of 19), one GATC mo-

Figure 1. Distribution of SeqA-binding sites (dots) in regions of bacteriophage l promoters (pR, pI and paQ) stimulated by the seqA gene prod- uct. The binding sites were determined using electron mi- croscopy; the data are taken from Słomińska et al. (2001, 2003a, 2003b). Nucleotide positions (from –250 to +250) are relative to transcription start points (+1; dotted vertical line) and are shown at the top of the panel.

MATERIALS AND METHODS

The list of genes whose transcription was ei- ther induced or repressed was taken from the work by Lobner-Olsen et al. (2003). Sequences of these genes (with adjacent regions), as well as of regions of randomly selected genes Figure 2. Distribution of GATC sequences (dots) in regions of promoters of genes for which de- whose transcription was reported to be creased (panel A) or increased (panel B) mRNA SeqA-independent (Lobner-Olsen et al., levels in the DseqA mutant were demonstrated re- 2003), were taken from the GenBank data- cently (Lobner-Olsen et al., 2003). base (http://www.ncbi.nml.nih.gov). The se- Nucleotide positions (from –250 to +250) are relative quences were aligned to show locations of to transcription start points (+1; dotted vertical line) GATC motifs relative to the transcription and are shown at the top of each panel. Quantitative start site of each promoter. data on the levels of particular transcripts are given in Lobner-Olsen et al. (2003).

RESULTS AND DISCUSSION tif occurs in a region near 100 (between 60 and 140) bp downstream of the promoter Results of our analysis of distribution of (promoters of genes: cydD, gltB, trpE, holD, GATC motifs in regions of promoters of hisG, gapC, asnA, dld, osmE), often followed E. coli genes activated, repressed and unaf- by at least one more such sequence located ei- fected in the E. coli seqA mutant are pre- ther downstream or upstream of the tran- sented in Figs. 2 and 3. Although it is difficult scription start site (all promoters listed above 944 B. Strzelczyk and others 2003 except those for genes cydD, trpE and asnA). SeqA binding proposed by Brendler & Austin A similar distribution of GATC motifs occurs (1999) on the basis of in vitro studies with downstream of the bacteriophage l pI pro- oligonucleotides, namely the presence of at moter that is stimulated by SeqA (S³omiñska least two GATC sequences located on the et al., 2003a). Moreover, there are two neigh- same DNA side and separated by no more boring GATC sequences between positions than 3 helical turns. However, it was demon- +150 and +200 in the regions of three addi- strated experimentally that when using lon- tional promoters stimulated by SeqA (pro- ger DNA fragments, SeqA can bind to GATC moters for fecI, dps and tpr). Such double regions distributed in a completely different GATC motifs were found downstream of the manner than that suggested by Brendler & SeqA-activated l pR and paQ promoters Austin (1999) as a requirement for SeqA– (S³omiñska et al., 2001, 2003a, 2003b). These DNA interactions (Słomińska et al., 2001; types of distribution of SeqA-binding se- 2003a; 2003b). Interestingly, many promot- quences are significantly less frequent in re- ers that are not significantly affected by SeqA gions of promoters that are repressed by have relatively many GATC motifs in their re- SeqA (Fig. 2B) or are SeqA-independent gions while others are devoid of such se- (Fig. 3). Therefore, one may speculate that quences. It is plausible that the location of the GATC motif(s) relative to a promoter, described above, results in stimulation of transcription initiation by SeqA. Nevertheless, other pro- moters, which do not reveal characteristic patterns of GATC motifs, might be depressed in seqA mutants. There are several possible reasons that could explain this phenomenon. First, SeqA-dependent regulation of tran- scription due to changes in nucleoid struc- ture, suggested by Lobner-Olsen et al. (2003), cannot be excluded. Second, the regulation of transcription of some genes by SeqA may be Figure 3. Distribution of GATC sequences (dots) indirect, due to putative SeqA-mediated con- in regions of randomly chosen promoters of trol of expression of other genes, whose prod- genes for which mRNA levels in the DseqA mutant were found to be unaffected or changes relative ucts might be involved in transcription of to wild-type bacteria were less than two-fold (on those showed in Fig. 2A and 2B (note that in the basis of the results reported by Lobner-Olsen this figure, only genes whose transcript levels et al., 2003); in fact, most E. coli genes belong to differed between the wild-type and DseqA this group. strains at least two times are shown, and even Nucleotide positions (from –250 to +250) are relative minor changes in expression of genes coding to transcription start points (+1; dotted vertical line) for regulatory may have significant and are shown at the top of the panel. effects on expression of other genes, con- trolled by these proteins). Third, although such patterns of GATC motifs are favorable only a few examples of specific SeqA-medi- for formation of SeqA-DNA complexes able to ated activation of promoters have been re- stimulate transcription from the nearby pro- ported to date, it is clear that there are vari- moters. It is worth noting that these GATC ous mechanisms of regulation of transcrip- motifs found near the SeqA-stimulated pro- tion by this protein. For example, bacterio- moters generally do not follow the rules for phage l pR promoter seems to be directly acti- Vol. 50 SeqA-mediated transcription regulation 945 vated by SeqA (Słomińska et al., 2001; REFERENCES 2003b), whereas seqA gene product stimu- lates the activity of pI and paQ promoters by Boye E, Stokke T, Kleckner N, Skarstad K. facilitating action of another transcription ac- (1996) Proc Natl Acad Sci U S A.; 93: tivator, the CII protein (Słomińska et al., 12206–11. 2003a). Therefore, various locations of GATC Brendler T, Austin S. (1999) EMBO J.; 18: motifs may be necessary for different mecha- 2304–10. nisms of SeqA-mediated transcription stimu- Kang S, Lee H, Han JS, Hwang DS. (1999) J lation. We conclude that it is likely that for di- Biol Chem.; 274: 11463–8. rect stimulation of a promoter by SeqA, the presence of a GATC motif (followed by an- Lobner-Olsen A, Marinus MG, Hansen FG. other such motif) in the +100 region, or a cou- (2003) Proc Natl Acad Sci U S A.; 100: ple of GATC sequences in a region between 4672–7. +100 and +200, is required. Activation of Lu M, Campbell JL, Boye E, Kleckner N. (1994) other promoters shown in Fig. 2A is perhaps Cell.; 77: 413–26. indirect, for instance due to changes in nu- Skarstad K, Lueder G, Lurz R, Speck C, Messer cleoid structure. W. (2000) Mol Microbiol.; 36: 1319–26. The results of the microarray analysis per- formed by Lobner-Olsen et al. (2003) have one Slater S, Wold S, Lu M, Boye E, Skarstad K, Kleckner N. (1995) Cell.; 82: 927–36. more implication. It was demonstrated previ- ously that the properties of cellular mem- Słominska M, Węgrzyn A, Konopa G, Skarstad branes are significantly changed in seqA mu- K, Węgrzyn G. (2001) Mol Microbiol.; 40: tants (Węgrzyn et al., 1999). One possibility 1371–9. was that expression of genes coding for major Słomińska M, Konopa G, Ostrowska J, membrane proteins, or genes whose products Kędzierska B, Węgrzyn G, Węgrzyn A. are involved in production or degradation of (2003a) Mol Microbiol.; 47: 1669–79. other membrane compounds, is affected in Słomińska M, Konopa G, Barańska S, Węgrzyn the absence of active SeqA. However, there G, Węgrzyn A. (2003b) J Mol Biol.; 329: are no such genes among those significantly 59–68. stimulated or repressed by SeqA (Fig. 2). Therefore, other mechanism(s) must be re- von Freiesleben U, Rasmussen KV, Schaechter M. (1994) Mol Microbiol.; 14: 763–72. sponsible for the changes in cell membranes observed in seqA mutants. Węgrzyn A, Wróbel B, Węgrzyn G. (1999) Mol Gen Genet.; 261: 762–9. Wold S, Boye E, Slater S, Kleckner N, Skarstad K. (1998) EMBO J.; 17: 4158–65.