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Journal of Bacteriology, September 2005, p. 6403-6409, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6403-6409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Homologies and Divergences in the Transcription Regulatory System of Two Related Bacillus subtilis Phages

Laura Pérez-Lago, Margarita Salas, and Ana Camacho^*

Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain

Received 1 April 2005/ Accepted 13 June 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transcription regulation relies on the molecular interplay between the RNA polymerase and regulatory factors. Phages of the 29-like genus encode two regulatory proteins, p4 and p6. In 29, the switch from early to late transcription is based on the synergistic binding of proteins p4 and p6 to the promoter sequence, resulting in a nucleosome-like structure able to synergize or antagonize the binding of RNAP. We show that a nucleosome-like structure of p4 and p6 is also formed in the related phage Nf and that this structure is responsible for the coordinated control of the early and late promoters. However, in spite of their homologies, the transcriptional regulators are not interchangeable, and only when all of the components of the Nf regulatory system are present is fully active transcriptional regulation of the Nf promoters achieved.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria exert control of gene expression primarily at the level of transcription initiation using diverse mechanisms, which include sigma factors that confer specificity to the RNAP for certain set of promoters, and transcription factors or regulators that either help or hinder RNAP-promoter interactions (7, 22). Regulatory proteins were originally classified as activators or repressors if they enhanced or inhibited transcription, respectively. More recent studies, however, indicate that both activators and repressors can exert dual functions, activating or repressing transcription depending on where they bind to the DNA (2, 12, 20, 21, 27, 44, 47, 50). Moreover, most transcriptional regulatory systems rely on the function of more than one regulatory protein.

A fundamental question of gene regulation is how functional interaction between the proteins regulates differential gene expression when two or more transcriptional factors are involved. Studies of promoter regulation by multiple regulators indicated that both antagonism (23, 45) and synergism between regulators occurs. In Escherichia coli, synergy between cI and cyclic AMP receptor protein (CRP) (24), FIS and IHF, CytR with CRP, or HU with GalR has been documented at the acsP2 (6) crp (17), fis (34), bgl (11), proP (29), ropH (25), and gal (26) promoters. Other examples of synergy between factors rely on the formation of a higher-order structure involving cooperative binding of factors such as MalT and MelR with CRP in the activation of the malE and melAB promoters, respectively (19, 40, 49).

In Bacillus subtilis, synergistic binding to the DNA of two transcription regulators, proteins p4 and p6, results in the switch from early to late transcription of phage 29 (8, 9, 13). Protein p4 specifically binds to two regions of the phage genome, one placed between the A2c and A2b early promoters and the other between the early A2b promoter and the A3 late promoter (Fig. 1B) (10, 41). Protein p6 is a dimeric histone-like protein which binds to the DNA, giving rise to an extensive DNA-protein complex (1, 46). The transition from early to late transcription relies on the formation of a p4-p6 multimeric complex able to antagonize the RNAP at promoters A2c and A2b and concomitantly synergize the binding of RNAP to the late A3 promoter.

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FIG. 1. A. Schematic representation of the transcription map of the genomes of phage 29 and Nf. The scheme shows gene organization and the locations of the early promoters A2c and A2b and late promoter A3, with arrows indicating the direction of transcription. The phage terminal protein (TP) is shown attached to the 5' ends of the genome. B. Comparison of phage Nf and 29 nucleotide sequences of the region including promoters A2b, A2c, and A3. The –10 and –35 elements for B. subtilis A-RNAP are indicated. Promoter start sites are denoted by arrows with the head toward the direction of transcription. Binding sites of phage 29 protein p4 are indicated, and the inverted repeats at each site in phages 29 and Nf are denoted by arrows.

The 29-like genus of phages has been subclassified into three groups based on serological properties, genomic maps, and DNA sequences (38, 51). The first group includes phages 29, PZA, 15, and BS32; the second group includes B103, Nf, and M2Y; and the third group contains only phage GA1. All three groups have similar genome and promoter organization (30, 35), and all of them encode proteins homologous to 29 proteins p4 and p6. We have studied the proteins and molecular mechanisms involved in the repression of the early promoters A2c and A2b and in the activation of the late promoter A3 in the phylogenetically related phage Nf. The results indicate that, in spite of the similarity of genomic organization and the functional conservation of the proteins involved in transcription regulation, the DNA sequence and proteins p4 and p6 have been optimized for the regulation of transcription of the Nf genome by a subtle divergent evolution.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proteins and nucleotides. B. subtilis RNAP was purified as described (48). Gene 4 of phage Nf, which encodes protein p4, was amplified by PCR and cloned in the Escherichia coli expression plasmid pT7.7. Bacteria harboring the Nf gene 4 were selected and checked by sequencing. The Nf protein p4 was expressed after induction with isopropylthiogalactopyranoside (IPTG) following standard procedures (43) and purified as described (3). Protein p6 of phage 29 and Nf, and protein p4 of phage 29 were purified as described (3, 37). Protein p4 and p6 concentration were determined as dimers (1, 31). Unlabeled nucleoside triphosphates and deoxynucleoside triphosphates were obtained from Pharmacia. [-³²P]ATP (3,000 Ci/mmol) and [-³²P]UTP (3,000 Ci/mmol) were purchased from Amersham International.

DNA substrates. The central promoter region of phage Nf was obtained by PCR amplification using the oligonucleotides Nf1, 5'-CATATTTTCCTTCTCTTCC-3', and Nf2, 5'-CTTCACGAAAATCTCGTTCCATCGG-3'.

The amplified DNA fragment was cloned at the SmaI site of a pUC18 derivative with transcriptional terminators at both ends of the plasmid polylinker. The plasmid contains the Nf sequence of promoters A2c, A2b, and A3 from position +68 of promoter A2c start point to position +60 of promoter A3 start point and was named pUC-NfLP.

The DNA fragments used in band-shift and runoff assays were obtained by PCR amplification of the Nf sequence of plasmid pUC-NfLP using the oligonucleotides Nf3, 5'-CATATTTTCCTTCTCTTCCTTTTCCTTCTCTTTC-3', and Nf4, 5'-CTCGTTCCATCGGCATATAGCCCATCTCCTTTC-3'.

The DNA fragments used in DNase I footprinting assays were obtained by PCR amplification of the Nf sequence of plasmid pUC-NfLP using the oligonucleotides Nf5, 5'-GTGTAGTAGGTAACGCCTTACTACTCTTTTAGTATATCATG-3', and Nf6, 5'-CAATAGATTATATTACTATTTTATTATATGACGGAAACCC-3'.

For band-shift assays and DNase I footprints, one of the primers was labeled by treatment with polynucleotide kinase and [-³²P]ATP for 45 min at 37°C prior to amplification. Each fragment was purified by NuSieve GTG agarose (FMC) gel electrophoresis and QIAGEN gel extraction kit.

Band-shift assays. Binding reactions (20 µl) contained the corresponding 5'-end-labeled fragment of Nf DNA, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 100 mM KCl, 1 µg of poly(dI-dC), and 10 µg of bovine serum albumin. Proteins p6 and p4 were added in the amounts indicated and incubated for 20 min at 4°C. After addition of 2 µl of 30% (vol/vol) glycerol, samples were loaded onto a nondenaturing 6% polyacrylamide gel. Electrophoresis was run at 25 mA/gel for 3 h at 4°C. Gels were dried and quantified by using a Fuji Bas-IIIs Image analyzer.

DNase I footprinting. Footprint reactions contained end-labeled DNA, 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl₂, 1 µg of poly(dI-dC), 1 mM EDTA, and 1 µg of bovine serum albumin in a final volume of 20 µl. Proteins p4, p6, and RNAP were added in the amounts indicated and incubated for 20 min at room temperature. DNase I footprinting was performed with 0.05 U of RQ1 DNase I (Promega) at 37°C for 2 min (16). Reactions were stopped by adding EDTA (10 mM) and 10 µg of tRNA. To determine the positions of the nucleotides in the DNA fragments and as a control for molecular weight, G+A sequencing of each fragment was carried out following the method of Maxam and Gilbert (28) and the reaction was run in parallel with the DNase I reactions. Digestion products were analyzed by electrophoresis on 6% polyacrylamide gels in the presence of 8 M urea.

In vitro transcription assay. Runoff transcription assays (20 µl) contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM dithiothreitol, 2 µg of poly(dI-dC), 100 mM KCl, 10 U of RNasin, 100 µM each GTP, ATP, and CTP, 100 µM [-³²P]UTP (1 µCi), and 2 nM DNA template. The template fragment contained promoters A2c, A2b, and A3. RNAP was added and, after 20 min at 37°C, reactions were stopped by addition of 0.15% sodium dodecyl sulfate and 2.5 mM EDTA. Transcripts were analyzed by electrophoresis in 6% denaturing polyacrylamide gels. Quantification of the transcripts was carried out by using a Fuji Bas-IIIs Image analyzer.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phages 29 and Nf, classified in different evolutionary groups, have similar promoter organization. Three promoters, two of them homologous to 29 early promoters A2b and A2c and the other homologous to 29 late promoter A3, could be distinguished in the Nf genome (Fig. 1A). In vitro transcription allowed definition of their transcription start sites (35). As in the case of the 29 promoters, the Nf promoters A2b and A2c contain consensus hexamers centered at positions –10 and –35, while the –35 element of promoter A3 has a very poor match to the consensus (Fig. 1B). Four binding sites for 29 p4 (p4) were defined on 29 DNA: sites 1 and 2, separated by 1 bp, are located between promoters A2c and A2b, while sites 3 and 4, also separated by 1 bp, are located between promoters A2b and A3 (10). A p4 binding site consists of a 19-bp AT-rich sequence flanked by the sequence 5'-TXG(A/T)_3-3' (4, 36, 39). Nf has only two sequences homologous to the p4 binding site positioned with respect to its promoters, as in the case of 29 sites 1 and 3. Phage Nf has proteins homologous to p4 (Fig. 2) and p6 (14), which in 29 are involved in the coordinated regulation of promoters A2c, A2b, and A3.

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FIG. 2. Amino acid sequence of the proteins from phage 29 and Nf. Identities are shown as dots. The total number of amino acids is in parentheses. The sequence of protein p4 from our strain of phage Nf (Nf a) differs in the amino acids at positions 30 and 86 from the sequence (Nf b) published previously (33).

To investigate the molecular mechanism underlying the regulation of Nf promoters A2c, A2b, and A3, we first analyzed the interaction of the homologous Nf proteins p4 (p4N) and p6 (p6N) with the Nf sequence including promoters A2c, A2b, and A3 by band-shift assays. Binding of protein p4N resulted in two main electrophoretically different complexes (I and II), which agree with the presence of two putative binding sites and might correspond to p4N attached to one or both sites of the DNA fragment (Fig. 3A, lane b). Protein p6N binding resulted in a retarded DNA band (Fig. 3A, lane d). To study functional homologies between the Nf and 29 proteins, we assayed in parallel the ability of p4 and p6 to interact with the Nf sequence. Binding of p4 gave rise to only one retarded band, indicating that, if p4 binds to the Nf sites 1 and 3 as does p4N, either the complexes are electrophoretically identical, or only one of them was stable in this assay (Fig. 3B, lane b). The protein p6-DNA band had a slightly slower mobility than free DNA (Fig. 3B, lane d).

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FIG. 3. Band-shift assay of the binding of p4 and p6 to the DNA fragment containing Nf promoters A2c, A2b, and A3. The p4 and p6 nucleoprotein complexes were analyzed in native polyacrylamide gels. Proteins were added according to the scheme above the autoradiograms, where p4 and p6 are the proteins of phage 29 and p4N and p6N are the phage Nf proteins. A. Complex formation with the Nf proteins. The concentration of p4N used was 80 nM. The concentration of p6N at lanes c and d was 8 and 16 µM, respectively, and at lanes e to h was 2, 4, 8, and 16 µM, respectively. B. Complex formation with the 29 proteins. The concentration of p4 used was 80 nM. The concentration of p6 at lanes c and d was 8 and 16 µM, respectively, and at lanes e to h was 4, 8, 16, and 32 µM, respectively. Nucleoprotein complexes are indicated at the side. The DNA fragment includes from nucleotide +67 of promoter A2c start site to position +48 of promoter A3; this sequence encloses sites 1 and 3.

In the band-shift assay, when p4N and p6N were added together, a unique band was obtained (Fig. 3A, lanes e to h). At the lower p6N concentration (lane e) the mobility of the complex is slower than that of the p6N/DNA complex or the p4N-DNA I complex but slightly faster than that of the p4N-DNA II complex and there is an increase in the mobility of the p4N-p6N-DNA complex as larger amounts of p6N are added (lanes e to h), which might suggest modification of the complex shape or of the net ion charge.

In 29, proteins p4 and p6 bind synergistically to the sequence including promoters A2c to A3. Simultaneous binding of proteins p4N and p6N to the Nf sequence shows complex formation at p6N concentration more than fourfold lower than required when p6N was assayed alone (Fig. 3A, see lanes c and e), indicating synergistic binding. In contrast, when the 29 proteins were tested with the Nf sequence, p4/p6 complexes were formed (Fig. 3B, lanes g and h), but eightfold more protein p6 was required and less than 50% of the DNA was complexed with p4 and p6. Hence, p4 and p6 do not bind synergistically to the Nf sequence. At the lower concentration of p6 (lane e), three bands were obtained which might correspond to p6-DNA, p4-DNA, and p4-p6-DNA complexes in view of the fact that increasing the amount of p6 caused the faster moving band to disappear, whereas the amount of the slower band increased.

Binding of p4N and p6N to Nf DNA was further analyzed by nuclease protection experiments. Figure 4 (lane b) shows changes in the digestion pattern between nucleotides –20 and –60 and between positions –118 and –152 relative to the promoter A2c start site in the presence of p4N. These DNA regions correspond in position to binding sites 1 and 3 described in the 29 genome (Fig. 1B). Binding of p4N to the sequence homologous to site 1 is reflected by protections at positions –20 and –22 and from –38 to –56, and hypersensitivities at positions –23 and –35 relative to the A2c promoter transcription start site (Fig. 4, lane b). Binding of the protein to the sequence homologous to site 3 induced hypersensitive bands at positions –129 and –139 and increasing protection of the intervening area (Fig. 4, lane b).

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FIG. 4. Autoradiogram of the DNase I footprinting of proteins p4 and p6 bound to Nf DNA. The fragment contains the Nf sequence from position +24 of the promoter A2c start site to position +16 of the promoter A3 start site and was labeled at the upper DNA strand of Fig. 1. Proteins present in each assay are indicated at the top of each lane, where p4 and p6 are the proteins of phage 29, and p4N and p6N are the phage Nf proteins. Sequences protected by the p4 proteins from the nuclease are indicated by vertical bars at the left. Distances in bp from the promoter A2c transcription start site are indicated. Protein p4 was added at 700 nM. Protein p6N was added at 10 and 20 µM in lanes d and e, respectively, whereas p6 was at 15 and 20 µM in lanes f and g, respectively. Where increasing concentrations of p6 were used, 2.5, 5, 10, 15, and 20 µM p6N and 5, 10, 15, and 20 µM p6 were added.

The footprint pattern of p4 was similar to that of p4N (lane c). Taking into account the DNA sequence homology and the p4-mediated protection patterns, we define here two p4N binding sites, 1 and 3, at the Nf sequence. Hence, there is binding homology between p4N and p4, but, while four p4-binding sites were characterized in phage 29, only sites 1 and 3 are apparent in phage Nf. Analysis of p6N binding shows essentially the same pattern of digested bands in the absence and in the presence of 10 µM of protein (Fig. 4, lane d). However, a protection pattern along the entire fragment was obtained when 20 µM of p6N was added (Fig. 4, lane e). When binding of p6 to the Nf fragment was assayed, more protein was required to obtain a pattern similar to that of p6N (Fig. 4, lanes f and g, and data not shown).

In phage 29, proteins p4 and p6 form an extended nucleoprotein complex covering the sequence from promoter A2c to promoter A3. Analysis of the p4-p6 complex formed at the Nf DNA with homologous and heterologous proteins was carried out by DNase I footprinting (Fig. 4). From the results obtained, several conclusions can be reached. First, in the presence of p4N and p6N a nucleoprotein complex is formed along the sequence analyzed. On the basis of the protections and hypersensitivities to DNase I elicited, it seems that, in the complex, protein p4N remains bound to sites 1 and 3 (Fig. 4, lanes h to l). There is synergy between p4N and p6N, since more p6N was needed to detect binding when the protein was added alone than when p4N was present (>10 µM versus 2.5 µM) (Fig. 4, compare lanes d and h). Increasing the amount of protein p6N to 15 µM or higher results in the extension of the nucleoprotein complex beyond p4N binding sites 1 and 3 (lane k). When nucleoprotein complex formation was assayed with the 29 proteins, a similar pattern was obtained (Fig. 4); however, the complex was formed less efficiently. On the basis of the protection patterns, an eightfold higher p6 concentration was needed when p6 and p4 were compared to their Nf counterparts (Fig. 4, compare lanes h and m), in agreement with the band-shift experimental data.

As shown in Fig. 5, the RNAP binds to promoter A2c as reflected by modifications of the DNase I pattern from positions –59 to –9 but poorly recognizes promoter A3 (compare lanes g and h). Binding of RNAP to promoter A2b was not detected by this technique. Formation of the p4/p6 complex occupying the sequence including promoters A2c, A2b, and A3 suggests that the B. subtilis ^A-RNAP may compete for the promoter sequence. We assayed this hypothesis in competition experiments where the p4N/p6N nucleoprotein complex was preformed and then competed by increasing amounts of RNAP (Fig. 5, compare lane h with j to m). The results show that (i) without RNAP, the footprint of the p4N/p6N complex extended beyond p4 binding sites 1 and 3 (lane i); (ii) with 14 and 28 nM RNAP, there are modifications of the protection pattern at positions –186 to –163 relative to promoter A2c start site, which correspond to positions –32 to –55 of promoter A3 start site (lanes j and k); and (iii) RNAP-derived protection at promoter A3 increases toward the main promoter core when concentrations of RNAP higher than 28 nM were used (lanes l and m). Protections between positions –32 and –37 may correspond to /DNA interactions, while protections between positions –38 and –45 and positions –49 and –55 might be due to alpha-C-terminal domains (CTDs) binding (10, 42). Hence, the p4N-p6N complex stabilized RNP at promoter A3 which may be promoted by CTD-p4N interactions as it occurs in 29 (32). In contrast, the digestion pattern was not significantly modified at promoter A2c, except for the hypersensitivity at position –29, which seemed to diminish when the amount of the RNAP increased.

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FIG. 5. Analysis by DNase I footprinting of the competition between the RNAP and the p4/p6 nucleoprotein complex for binding to Nf promoters A2c, A2b, and A3. Binding of RNAP to the promoters in the presence of p4N (700 nM) and p6N (10 µM) was studied, adding 14, 28, 56, and 112 nM RNAP (lanes j to m and o to r, respectively). Controls with only proteins p4 (700 nM), p6N (10 and 15 µM), p6 (15 and 20 µM), or RNAP (56 nM) were carried in parallel. The DNA fragment includes from nucleotide +24 of the promoter A2c start site to +16 of the promoter A3 and was labeled at the lower strand. Promoters A2b, A2c, and A3 are depicted at the left. Several positions relative to the promoter A2c start site are indicated to the right and several positions relative to the promoter A3 start site are in parentheses. Protein p4 binding sites 1 and 3 are indicated to the left.

With the aim to study the specificity of the p4/p6 complexes, competition assays between RNAP and the p4/p6 complex formed on the Nf sequence were carried out (lanes n to r). Using concentrations of proteins similar to the ones employed in the analysis of the p4N/p6N complex, the protection pattern indicates that the p4/p6 complex extended only from site 3 to site 1 (lane n). An increase in the RNAP concentration resulted in the progressive displacement of the p4/p6 complex (compare lanes n and r) with the appearance of RNAP bound to promoter A3, although the RNAP-mediated protections suggest that here only the CTD of the subunits are stably bound (lanes q and r).

The coordinated regulation of the Nf promoters was analyzed by in vitro transcription experiments in the absence or presence of the Nf proteins p4N and p6N. As shown in Fig. 6, the RNAP recognized and transcribed from promoters A2b and A2c. Transcription derived from promoter A3 was very low except when 80 nM p4N was added (lanes a and b); with 270 nM p4N partial repression of promoter A2c was in addition achieved (not shown). When transcription was carried out in the presence of p6N, the level of transcripts derived from promoters A2b and A2c were not greatly affected indicating that p6 does not interfere specifically with the expression of these promoters (lane d). Simultaneous addition of proteins p4N and p6N produced the shut-off of promoters A2b and A2c while promoter A3 remained activated (lane c). These results indicate that synchronized regulation of Nf promoters A2c, A2b and A3 requires the simultaneous presence of proteins p4N and p6N.

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FIG. 6. In vitro transcription from Nf promoters A2c, A2b, and A3 mediated by proteins p4 and p6, where p4N and p6N are the proteins of phage Nf and p4 and p6 are the phage 29 proteins. The template DNA used in these assays included from position +67 of the Nf promoter A2c start site to position +48 of promoter A3; hence, we expected RNAs of 67 nucleotides when derived from promoter A2c, 163 nucleotides from promoter A2b, and 48 nucleotides from promoter A3. The proteins used in each assay are indicated above the autoradiograph. The concentration of the proteins was: for p4, 80 nM; for p6, 4 µM; and for RNAP, 11 nM. The transcripts corresponding to each promoter are indicated at the right.

To provide insights into the functional activity of the regulatory system derived from each phage, the function of the homologous p4/p6 and heterologous p4/p6 complexes (p4N/p6 and p4/p6N) on the regulation of the Nf promoters was analyzed (Fig. 6, lanes e to i). In contrast to the results obtained with p4N and using the same concentration of proteins, protein p4, capable to activate the 29 promoter A3 (not shown), did not activate the Nf promoter A3 as p4N does (lane g) and was also incompetent for activation of promoter A3 either in the presence of p6N or p6 (lanes f and h). None of the p4/p6 complexes (except the p4N/p6N complex described above) repressed efficiently promoters A2c and A2b (lanes e, f, and h). Increasing the concentration of p4 more than 3-fold and in the presence of 10 µM p6, 2-fold activation of A3 and partial repression of promoters A2c and A2b were achieved (not shown). Protein p6 behaved like p6N does (lane i). Taking into account these data, it can be concluded that the concomitant presence of p4N and p6N confers specificity to the regulatory system.

Differences in the affinity of p4N and p4 for sites 1 and 3 might explain the functional differences observed on the regulation of the promoters. The analysis of the binding affinity of the p4 proteins to site 3 was carried out in band-shift assays with oligonucleotide-derived sites of 60 bp containing Nf site 3, flanked by nonspecific sequence. Comparison of the p4/DNA complex formed by p4N and p4 revealed that p4N binds about fivefold better than p4 (Fig. 7), which shows why only p4N was able to activate promoter A3 (Fig. 6).

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FIG. 7. Study of the binding of p4 proteins for phage Nf site 3 by band-shift assay. The DNA sequence used contains the 31 bp of site 3 surrounded by 15 bp of unspecific sequence. The p4 nucleoprotein complexes were analyzed in a native 6% polyacrylamide gel. Proteins (80 and 160 nM) were added according to the scheme above the autoradiograms, where corresponds to p4 of phage 29 and N corresponds to p4 of phage Nf.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We show here that in phage Nf, protein p4N is required for activation of promoter A3 and the additional presence of protein p6N results in the repression of the early promoters A2c and A2b. Binding of p4N and p6N to the region containing these promoters gives rise to a nucleoprotein complex covering from downstream of promoter A2c to downstream of promoter A3. The additional presence of RNAP results in the stabilization of the enzyme at promoter A3, while the enzyme does not seem to compete efficiently with the p4N-p6N complex bound along the sequence of promoters A2b and A2c. In phage 29, a lower concentration of p4 is required in vitro for activation of 29 promoter A3 than for repression of promoter A2c, and the coordinated regulation of promoters A2b, A2c, and A3 requires the presence of proteins p4 and p6 (13). Hence, phages 29 and Nf follow a common mechanism for the switch from early to late transcription.

The similarities at the level of promoter organization together with the existence of 78.6% identity between the p4 proteins and 50.5% identity between the p6 proteins (15) suggested common interactions between the molecules involved in this process: DNA, RNAP, p4, and p6, which could drive the functional substitution of the p4s and p6s, especially taking into account that p6 is a histone-like protein. However, our results when homologous and heterologous proteins were compared indicate that the regulatory systems of 29 and Nf have evolved in such a way that each one has been optimized for regulation of its cognate promoters.

Protein p4 does not activate Nf promoter A3 to the same extent as p4N does. Several factors could contribute to this fact. On the one hand, p4N binds about fivefold better to Nf site 3 than p4, which could be due to the fact that the proteins differ in one of the residues (Q5) involved in DNA binding (D. Badía, A. Camacho, L. Pérez-Lago, C. Escandón, M. Salas, and M. Coll, unpublished results). The nucleotide sequence of site 3 could also contribute, especially since Nf site 3 is 1 bp longer than the consensus sequence. On the other hand, protein p4 binds to its own DNA at tandem sites and the Nf sequence contains only two separated binding sites. Stable binding of p4 might require further stabilization of the p4-DNA complex through interactions between neighboring p4 dimers, while this stabilization energy would be superfluous when p4N binds to its own sequence.

The results presented here on the regulation of transcription support the hypothesis of specific protein-protein interaction between p4 and p6 and specific interaction also between p6 and DNA. None of the combinations of protein p4 with its homologous or heterologous p6 was able to encompass regulation of promoters A2c, A2b, and A3 except for the pair p4N-p6N. There was synergism between p4N and p6N when binding to the Nf sequence, and also synergistic binding of p4 and p6 to its cognate DNA sequence was described (9). However, even if the DNase I pattern of the 29 and Nf pairs were similar (Fig. 4), p4 and p6 do not bind synergistically to Nf DNA. These data indicate that protein p4 specifically recognizes the homologous protein p6 and vice versa, and a precise positioning of the proteins in the DNA is required for interaction. In agreement with these findings, studies on DNA replication with the phage 29 and Nf components had shown that the initiation of replication is selectively stimulated only when every component belonged to the same phage (5, 15, 18).

In conclusion, even if the regulation of transcription of 29 and its related phage Nf is similar, the regulatory factors p4 and p6 do not interchange efficiently. The data obtained suggest that the nucleoprotein complex p4N/p6N has evolved to be functionally adapted for the coordinated regulation of the Nf promoters.

 
We are indebted to J. M. Lázaro and L. Villar for proteins purification.

This work was funded by grant 08.2/0026.1/2003 from Comunidad Autónoma de Madrid to A.C., grant BMC 2002-03818 from the Spanish Ministry of Science and Technology to M.S., and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular "Severo Ochoa." L.P.-L is the holder of a predoctoral fellowship from Comunidad Autónoma de Madrid. The "Ramón y Cajal" program of the Spanish Ministry of Science and Technology supported A.C.

 
* Corresponding author. Mailing address: Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. Phone: 34-91 497 8434. Fax: 34-91 497 8490. E-mail: acamacho{at}cbm.uam.es.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, September 2005, p. 6403-6409, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6403-6409.2005
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This article has been cited by other articles:

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