Specific Binding of the Tray Protein to oriT and the Promoter Region for the tray Gene of Plasmid RlOO*

The tray gene product of plasmid RlOO was purified as a hybrid protein, Tray-collagen+galactosidase. The hybrid protein as well as the Tray’ protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among RlOO-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the Tray protein has a role as a component of endonuclease in recognizing its own oriT sequence. however, the hybrid and the Tray’ protein were different AT-rich se- quences (each 24 pairs in length) the tray

The tray gene product of plasmid RlOO was purified as a hybrid protein, Tray-collagen+galactosidase. The hybrid protein as well as the Tray' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT.
The oriT region consists of highly conserved and nonconserved regions among RlOO-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the Tray protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the Tray' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the tray gene (here called sbyB and sbyC). This suggests that the Tray protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the Tray protein.
Mg2+ is not required for the specific binding of the Tray protein.
Plasmid RlOO confers conjugal transfer ability on its host, a process in which DNA is transmitted from one bacterial host to another. RlOO and several other conjugative plasmids, such as F and Rl, share homology in the tra region which is responsible for DNA transfer (Sharp et al., 1973) and are considered to have the same conjugation system. The tra region consists at least 26 genes which are organized into three main operons, traM, trd, and tray-2 (for recent reviews, see Ippen-Ihler and Minkley, 1986;Willetts and Skurray, 1987). Transcription of the tray-2 operon is positively regulated at the promoter Pyz by the product of traJ (Gaffney et al., 1983;Fowler et al., 1983;Mullineaux and Willetts, 1985;Fowler and Thompson, 1986;Finlay et al., 1986a;Inamoto et al., 1988).
One of the initial events in DNA transfer upon expression of the tra genes is the strand-and site-specific nicking at the origin of transfer, oriT, by a plasmid-specific endonuclease (Everett and Willetts, 1980 region (Willetts and Maule, 1986). oriT sequences of RlOO and related plasmids (F, Rl, ColB4, and P307) have been determined (Thompson et al., 1984;Ostermann et al., 1984;Finlay et al., 1986b;McIntire and Dempsey, 1987;GGldner et al., 1987;Inamoto et al., 1988). The oriT region contains a highly conserved region as well as a nonconserved region among these plasmids, although the minimal oriT region is not clearly defined as yet. DNA nicking is considered to occur at several sites within the conserved region immediately adjacent to the nonconserved region (Thompson et al., 1984(Thompson et al., , 1989.
In this paper, we show that the tray gene product specifically binds to the nonconserved region in oriT as well as to the promoter region Pyz preceding the tray gene. We consider the possibility that the Tray protein has a dual role: to recognize its own oriT sequence as a component of a presumptive endonuclease responsible for nicking at oriT and to regulate the expression of its own gene.
&187-XEl.  Inamoto et al. (1988). Plasmids pYY5 (Yoshioka et al., 1987) and pYY30-1 (Yoshioka, et al., 1990) carried the WI-D fragment and G fragment of RlOO, respectively. The structures of several of these plasmids are shown in Fig. 2. Media-Culture media used were LB broth, L-rich broth, and &medium (Yoshioka et al., 1987). b-medium was used for transformation of plasmid DNA (Yoshioka et al., 1987 et al. (1978). The crude lysis method (Machida et al., 1982) was used to prepare a small amount of plasmid DNAs from large numbers of cell cultures. An alkaline lysis method (Maniatis et al., 1982;Yoshioka et al., 1987) was used to prepare plasmid DNA for nucleotide sequencing, construction of recombinant plasmids, and gel retardation assays. Purification of the Hybrid and TruY' Proteins-The hybrid protein was purified according to the procedure described by Inamoto et al. (1988) with some modifications; the membrane fraction was removed by centrifugation at 100,000 x g for 1 h; a P-galactosidase-specific affinity column and columns PD-10 (Pharmacia LKB Biotechnology Inc.) were used at 6 "C. The purified hybrid protein was dialyzed at 0 "C against one change of buffer B-G50, or one change of buffer B, containing 10% (v/v) glycerol (here called buffer B-G10). The hybrid protein in buffer B-G10 was frozen in dry ice and stored at -80 "C, and the protein in buffer B-G50 was stored at -20 "C. The Tray' protein was purified at 4 "C! as follows. for 5 min at is"C. Two bl of the'hybiid or Tray' protein in buffer B-G10 was added and incubated for 20 min at 28 "C. Then, 0.5 ~1 of 10 mg/ml heparin was added, and the mixture was further incubated for 10 min. The samples were loaded onto a 4 or 5% polyacrylamide gel (2 mm thick) in TAE buffer and electrophoresed at 12.8 V/cm at 4-6 "C.
The 5'-ends of the fragment were labeled with 32P and cleaved with RsaI, and the XhoII-RsuI fragment was purified as described above. To prepare the 322-bp AccI-AuuI fragment labeled at the AuuI site, pSI87-HE ( Fig. 2C) was digested with AuaI which cleaves the plasmid at one site, labeled with 32P at the 5'-ends of the DNA, and then cleaved with AccI. To prepare the 322-bp AccI-AuuI fragment labeled at the AccI site, pSI87-ES1 was digested with AccI which cleaves the plasmid at one site, labeled with 32P at the 5'-ends of the DNA, and cleaved with AuaI. These "P-labeled fragments were purified as described above. DNase Z Footprinting Experiment-TE buffer (2 ~1) containing the 32P-labeled fragment (approximately 0.1 pmol) was mixed with 53 pl of distilled water and 20 ~1 of 5 X binding buffer E' and preincubated for 5 min at 28 "C. Then, 20 ~1 of buffer B-G10 containing the hybrid protein (0 -16 pg) or the collagenolyzed hybrid protein (0 -15 fig) was added, and the mixture was incubated for 20 min at 28 "C. After the addition of 5 ~1 of 10 mg/ml heparin, the mixture was further incubated for 10 min. The sample was treated with DNase I (0.01 -0.02 unit) for 2 min under the condition described by Galas and Schmitz (1978). The DNA chains were separated on 8% and 6% polyacrylamide sequencing gels containing 8 M urea. Markers were prepared by the dideoxynucleotide method (Messing, 1983;Sanger et al., 1977). We used a sequencing kit (Takara Shuzo Co.), DNA Dolvmerase I (Klenow), and a primer whose 5'-end was labeled with3'P usingpolynucleotide kinase-and [y-32P]ATP. Primers used were 5'-ACTGATCCTAATAAGAGTCG-3' (ScuI primer). 5'-GATCCCATTTATAAACATCA-3' (XhoII primer), 5'-ATACATT-CAAAACATGATTT-3' (AccI primer), and 5'-CCGGGCACCTAA-GAGCTTG-3' (AuuI primer). These primers were synthesized using a DNA synthesizer 380B (Applied Biosystems).

RESULTS
Tray Protein and Its Binding Specificity to the oriT Region-The tray gene product was purified as a hybrid protein with collagen+galactosidase (LacZ) (Fig. IA; Inamoto et al., 1988). The hybrid protein contains a 74-amino-acid sequence of the tray gene product (75 amino acids) whose C-terminal Leu residue was substituted by the collagen-LacZ sequence (Inamoto et al., 1988  The smaller one (9.3 kDa) was close to the expected molecular mass of the Tray portion (8.8 kDa), in which the C-terminal mixed with the hybrid protein or the Tray' protein, and then Leu residue in the native tray gene product was presumably subjected to gel electrophoresis. In the presence of heparin, displaced by Pro-Gly-Pro-Val.
Although this protein (we call only the 286-bp XhoII-RsaI fragment containing the oriT it Tray') could be purified to homogeneity using a cation region migrated slowly from its original position and formed exchange column (data not shown; see Materials and Metha retarded band in a polyacrylamide gel (Fig. 3A, lanes 2 and ods), we used the mixture containing Tray' and LacZ portions 4), indicating that a complex was formed due to specific generated upon collagenolysis for the studies to be described binding of the proteins to the 286-bp fragment. Addition of below on the properties of the Tray' protein.
an excess of the proteins resulted in a mobility shift of all of To know whether or not the hybrid protein specifically the fragments (Fig. 3A, lane 3) or of some others in addition binds to the oriT region, the plasmid pSI87-XEl DNA con-to the 286-bp fragment (Fig. 3A, lane 5), representing nontaining oriT or the vector plasmid pUC19 DNA (Fig. 2) was specific binding of the proteins to DNA. Nonspecific binding mixed with the hybrid protein and subjected to gel electro-of the hybrid and Tray' proteins was also observed in the phoresis. Both samples migrated more slowly than the native absence of heparin (data not shown).
plasmid DNAs in an agarose gel (data not shown), suggesting The specific binding of the Tray' protein to the'286-bp that a complex was formed between each plasmid DNA and fragment was observed in the absence of MgZ+ or in the the hybrid protein. The complex of pSI87-XEl was, however, presence of 50 mM EDTA (Fig. 3B). The protein-DNA comresistant to heparin, while the complex of pUC19 was sensitive plex was formed inefficiently in the presence of 10 mM Mgr to heparin (data not shown). Both complexes were sensitive (Fig. 3B). Specific binding was observed in a pH range of LOto SDS to give rise to native plasmid DNAs in a gel (data not 8.5 and in buffers containing NaCl from 50 mM to 1.1 M (data shown). These findings suggest that the hybrid protein spe-not shown; see "Materials and Methods"). Specific binding of cifically and noncovalently binds to the oriT region carried the hybrid protein was also observed under the conditions by pSI87-XEl. described above (data not shown).
To further prove this suggestion, pSI87-XEl DNA was Tray Protein Binds to a 36-bp Sequence in the Noncondigested with the restriction enzymes RsaI and XhoII (Fig. 2), served Region in oriT-In order to identify the sequence  Fig. 4A, the hybrid protein as well as the Tray' protein protect the region (nucleotide positions 27 to 60 shown in Fig. 5A) within the nonconserved region among plasmids related to RlOO. Protection against DNase I digestion by the hybrid and Tray' proteins was also examined on the other strand of the fragment. Protection was observed for a region covering positions 28 to 61, as depicted in Fig.  5A. The protected sequence is very AT-rich (86% AT). Enhancement of cleavage by DNase I in the presence of the hybrid or Tray' protein was not observed on either strand (see Fig. 4A).
TraY Protein Also Binds to Pvz, the Promoter Region Preceding tray-To see whether or not the hybrid protein or the Tray' protein recognizes other parts of the tra region besides oriT, we carried out a gel retardation assay by two-dimensional gel electrophoresis. The DNA fragments generated by EcoRI and AccI double digestion of plasmid pSI28 containing the EcoRI fragment r4 (Fig. 2) were incubated with or without the hybrid protein and electrophoresed in the first dimension in an agarose gel (Fig. 6A). After treatment of the gel with SDS, electrophoresis was carried out in the second dimension. The fragments C and D from pSI28 (Fig. 2) formed several retarded bands seen as off-diagonal spots (see the brackets in Fig. 6B), indicating that these fragments are bound by the protein. Fragment C contains oriT, while fragment D contains Puz, the promoter region preceding the tray gene (Fig. 2). Using the restriction fragments generated from plasmid pSI87-HE carrying the Puz region (Fig. 2), the 173-bp DraI-StuI fragment, which was within the 322-bp region flanked by AccI and AuaI sites (see Fig. 2), was specifically bound by the hybrid and Tray' proteins. Restriction fragments generated by the plasmids, which carry EcoRI fragments (r3, r5, and r6) and SalI-D and G fragments containing different segments of tra of RlOO (see Fig. 2), were not bound by the hybrid protein (data not shown).  Fig. 5A). B, the 322-bp AccI-AuaI fragment containing the Puz region which was labeled with "P at the AuaI site (see Fig. 2 and "Materials and Methods").
Lanes l-6, samples containing the same amount of the hybrid protein as those used in lanes 1-6 in A-a, respectively; lanes 7-10, markers which were prepared by the dideoxynucleotide methods using the AuaI primer (see "Materials and Methods") in the presence of ddCTP, ddTTP, ddATP, ddGTP, respectively. The regions protected by the hybrid protein strongly and weakly are indicated by a filled box and a hatched box, respectively.
Locations of frames coding for t&and tray are indicated by open and closed arrows, respectively. Numbers indicate base positions (see coordinates shown in Fig. 5B).
To determine the sequence recognized by the hybrid protein within the 322-bp AccI-AuaI fragment containing Pyz, DNase I footprinting experiments were carried out. Strong protection was observed for the region at nucleotide positions 1637 to 1659 (Figs. 4B and 5B). Weak protection was also observed for the region at positions 1593 to 1615 (Figs. 4B and 5B). To obtain the same extent of protection in the latter as the former, over 100 times more hybrid protein has to be added (see Fig. 4B). Strong and weak protection was also observed on the other strand of the fragment, as depicted in Fig. 5B. Enhancement of cleavage by DNase I in the presence of the hybrid protein was not observed on both strands (see Fig.  4B). Similar results were obtained using the Tray' protein instead of the hybrid protein (data not shown). Both sequences strongly and weakly recognized by the hybrid protein and the Tray' protein were also AT-rich (88% and 76% AT, respectively) and were located in the Puz region. There is a homologous sequence, which matches 15 out of 19 base pairs, between the sequence bound by the hybrid and Tray' proteins in the oriT region and the sequence strongly bound by the Specific Binding of TraY of RlOO to oriT and Pm A. A, an ethidium bromide-stained agarose gel (1.6%) after electrophoresis in one dimension of PSI28 DNA (0.19 pg) digested with AccI and EcoRI. Lane 1, the DNA sample without the hybrid protein; lane 2, the sample with 26 pg/ml hybrid protein.
The sizes of restriction fragments (A, B, C, and D) are shown in kilobases. B, an agarose gel after electrophoresis in two dimensions. After electrophoresis in the first dimension as shown in A, the protein-DNA complexes in the gel were dissociated in situ and electrophoresed in the second dimension (see "Materials and Methods"). The off-diagonal spots indicated by brackets are due to formation of complexes between the hybrid protein and C or D fragment. In this experiment, complexes were formed in the presence of 14 pg/ml hybrid protein. and Puz. A, nucleotide sequence of the XhoII-RsaI (ScaI) fragment (McIntire and Dempsey, 1987;Inamoto et al., 1988). Conserved and nonconserved regions in oriT among plasmids related to RlOO are shown. Proposed nick sites in ori?' of F (Thompson et al., 1984;Thompson et al., 1989) and two putative IHF-binding sites (McIntire and Dempsey, 1987) are indicated by solid arrowheads and thick underlines, respectively. The putative amino acid sequence encoded by gene X is indicated. The region (named sbyA) protected against DNase I digestion by the hybrid protein or the Tray' protein on each strand is shown by a bold bracket. B, nucleotide sequence of the Pyz region flanked by the coding regions for traJ and tray (Inamoto et al., 1988). The Shine-Dalgarno sequence preceding tray is indicated by bold letters. The regions (named sbyB and sbyC) strongly and weakly protected by the hybrid or Tray' protein on each strand against DNase I digestion are shown by bold and thin brackets, respectively. In A and B, the repeat sequence, TAA(A/T)T, seen in the sby sites are indicated by half-arrows inside brackets; the inverted repeat sequences are indicated by arrows between the strands. C, homologous sequences seen among the sby sites. Nucleotide positions are numbered from the first base of the nonconserved region among plasmids related to RlOO (see text). B -2nd proteins in the Puz region, as shown in Fig. 5C. The sequences in oriT as well as in PYZ contain inverted repeat sequences (Fig. 5, A and C). The sequence in Pyz weakly bound by the hybrid or Tray' protein shows poor homology with those in the other two binding sequences (Fig. 5C). These three bind-ing sequences contain a "TAA(A/T)T" sequence motif, as shown in Fig. 5, A and B. DISCUSSION We have shown that the hybrid protein (TraY-collagen+galactosidase) and the Tray' protein with a modified Cterminal end of the Tray protein bind to specific sites in the oriT region and in the promoter region (Pyz) preceding the traY gene. We have also shown that the hybrid and Tray' proteins bind nonspecifically to DNA in the absence of heparin or even in the presence of heparin when an excess amount of the proteins is added. Such nonspecific binding is characteristic of essentially all DNA binding proteins. It has been reported that the replication initiator protein of plasmid R6K fused to collagen-/3-galactosidase and the initiator moiety obtained by collagenolysis of the hybrid protein show the same specific DNA binding activity as the native initiator protein Bastia, 1983, 1984). We believe therefore that the observed properties of the hybrid protein or the Tray' protein in this paper represent those of the actual product of the tray gene. We thus refer below to both the hybrid protein and the Tray' protein as the Tray protein.
As described under "Results," the Tray protein binds to a specific site in the plasmid-specific region immediately adjacent to the highly conserved region in oriT among plasmids related to RlOO. We named this site sbyA (for specific binding site of the Tray protein) (Fig. 5A). This result indicates that 10 out of 11 base pairs; Rl has one homologous sequence which shows 11 matches out of 12 base pairs. The coordinates of oriT of F are from Thompson et al. (1984), and those to Puz of F are from Fowler et al. (1983). The coordinates to oriT of Rl are from Ostermann et al. (1984), and those of PYZ of Rl from Finlay et al. (1986a). the Tray protein has a function in recognizing its own oriT sequence. Unexpectedly, the Tray protein also binds to two sites (we name these sbyB and sbyC, as represented in Fig.  5B) in the Pyz region. This suggests that the Tray protein may regulate the expression of its own gene.
sbyA consists of 36 base pairs, while both sbyB and sbyC consist of 24 base pairs. This difference in length may imply the difference in the number of protomers of the Tray protein involved in binding to each sby region. If so, the number of protomers bound to sbyA is 1.5 times as great as that bound to sbyB and sbyC. There are homologous sequences within the three regions recognized by the Tray protein (Fig. 5C). These sequences must play an important role in recognition by the Tray protein. These sequences contain the "TAA(A/T)T" sequence motif (Fig. 5, A and B). This motif might constitute a core sequence recognized by the Tray protein.
Do the oriT and Pyz regions in other plasmids related to RlOO contain the sequences which are recognized by their own Tray proteins?
As shown in Fig. 7, there are homologous sequences between the oriT and Pyz regions in each of these plasmids. It is quite likely that these sequences in each plasmid are recognized by their own Tray protein. This is based on the consideration that tra genes of RlOO-related plasmids have the same organization and function. The plasmid-specified endonuclease responsible for strandand site-specific nicking at oriT to initiate DNA transfer is thought to be a complex of the products of tray and truZ (Everett and Willetts, 1980). Recently, traZ activity was found to be dependent on the DNA sequence of trul encoding DNA helicase I (Traxler and Minkley, 1988). It is intriguing to speculate that the binding of Tray protein facilitates the ability of the TraI (or TraZ) protein to bind and nick at the highly conserved region. DNA helicase I activity of the TraI (or TraZ) protein unwinds the duplex DNA from a nick(s) to generate the single strand of the DNA that is transferred to the recipient.
The distance between the proposed nicking sites in oriT and sbyA is 59-74 base pairs (see Fig. 5A). Upon contact with the Tray protein, the TraI (or TraZ) protein may induce a change in DNA structure such as looping of the intervening region between the nicking sites and sbyA to interact with the region adjacent to or around the nick sites. It is known that transfer of RlOO and F from the mutant lacking IHF (integration host factor) is reduced (Dempsey, 1987;Gamas et al., 1987) and that there is a possible IHF recognition site in the nonconserved region in oriT at nucleotide positions 9 to 21 in Fig. 5A (McIntire and Dempsey, 1987), adjacent to sbyA. It is interesting to consider that IHF may promote the interaction of the TraI (or TraZ) protein with the Tray protein and with the highly conserved region in oriT.