VIROLOGY
i
87,788-795
(1992)
DNA Sequences
Necessary
for Packaging
CHIKARA HASHIMOTO Department
AND
Bacteriophage
HISAO FUJISAWA’
of Botany, Faculty of Science, Kyoto University,
Received October
T3 DNA
16, 199 1; accepted
December
Kyoto 606, Japan 27, 199 1
A recombinant plasmid, pUCEl-TR, carrying a target for processing of the concatemer joint (TR) and sequences to the left of the target (El), is efficiently packaged into transducing particles during T3 phage infection. Using this plasmid packaging/transduction system, the minimal sequences necessary for packaging of T3 DNA were determined. The TR sequence contains the targets for initiation cleavage and termination cleavage of concatemer processing (pacCR and pacCL, respectively). A plasmid lacking pacCL was packaged as efficiently as pUCE1 -TR but one deleted forpacCR was packaged at a very low efficiency, showing that pacCR is essential for production of transducers but that pacCL is dispensable. DNA from transducing particles carrying a recombinant plasmid lacking pacCL or pacCR had the same right or left end as T3 DNA, respectively, but its other end was not unique. In the absence of pacCL, packaging is initiated from the DNA end created by cleavage at thepacCR and terminated at any sequence after packaging a headful of DNA. In the absence of pacCR, packaging is initiated from the DNA end created by nonspecific, inefficient cleavage and terminated by cleavage at the pacCL after packaging a headful of DNA. A 23-bp segment flanking the site where the mature right end is formed was found to support efficient formation of transducing particles. A 53-bp sequence, including a consensus sequence for the promoter for T3 RNA polymerase, was a responsible element in the El sequence for packaging of plasmid DNA. Deletions of the 5’-upstream sequence of the promoter sequence from the left decreased the promoter and packaging activities in parallel, but with those of the 3’-downstream sequence from the right, the packaging activity was impaired before the promoter activity, indicating that transcription from the promoter is necessary but not sufficient for T3 DNA packaging. 0 1992 Academic Press, Inc.
INTRODUCTION
ture monomers are cut from the concatemer. In a previous paper (Hashimoto and Fujisawa, 1988), we showed that a recombinant plasmid carrying the concatemer joint (TR) sequence, including the terminally redundant sequence, and its left side flanking (El) sequences from T3 concatemeric DNA (pUCE1 -TR; Fig. 1, lane I), can be packaged into transducing particles. Transducing T3 particles contained head-to-tail oligomers of plasmid DNA with the same termini as those of mature T3 DNA. Thus, the T3 concatemer joint sequence in the plasmid is recognized for processing and packaging by T3 phage. However, the TR sequence alone, containing the cleavage sites for processing of concatemeric DNA (pacC), is not sufficient and the El sequence is additionally required for recognition by the packaging machinery (tentatively named pacB). From these results, we proposed that a bipartite structure of the sequence necessary for DNA packaging (pat signal), consisting of pacB and pacC, may be general for the sequences required for initiation cleavage in DNA packaging [Mu (George and Bukhari, 1981) P22 (Casjens and Huang, 1982), X (Feiss, et a/., 1982), and Pl (Sternberg and Coulby, 1987)]. In a defined in vitro system for packaging T3 DNA which is composed of purified proheads and packaging proteins (Fujisawa et al., 1990), plasmid DNAs containing T3 concatemer joints are cleaved at the left end of the terminally redundant sequence after being packaged leftward (Fujisawa eT
During the growth of most double-stranded DNA bacteriophages, phage DNA is synthesized as concatemers and packaged into head precursors, called proheads, as mature monomers are cut from the concatemer. The cutting of concatemeric DNA in viva is coupled to headful packaging (see reviews: Murialdo and Becker, 1978; Earnshaw and Casjens, 1980; Black, 1981). Cutting can be strictly sequence specific as in X (Wu and Taylor, 197 1), T3 (Fujisawa and Sugimoto, 1983), and T7 (Dunn and Studier, 1983). In the case of Mu (Bukhari et al, 1976), P22 (Casjens and Huang, 1982) Tl (Ramsey and Rittchie, 1983) or Pl (Sternberg and Coulby, 1987) cutting is initiated from a specific packaging sequence called pat and terminated at any sequence after packaging a headful of DNA. In the case of T4, heads can be filled without any apparent sequence specificity by a headful mechanism (Streisinger et a/, 1967). The termini of mature DNA from phage T3 contain the same 230 base pair sequences, called terminally redundant sequences. DNA is synthesized as concatemers in which unit-length molecules are joined together in a head-to-tail fashion through the terminally redundant sequences. During packaging of DNA, ma’ To whom reprint requests should be addressed. 0042.6822/92
$3.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
788
789
DNA SEQUENCES NECESSARY FOR PACKAGING BACTERIOPHAGE T3 DNA
a/., 1990). Therefore, the cleavage reaction in the in vitro system would correspond to the termination cleavage in vivo. Although T3 and related phage T7 DNAs can be discriminated during packaging, the termination cleavage is not specific between T3 and T7. From these results, we conclude that the packaging specificity resides in the initiation event(s), including the initiation cleavage, during DNA packaging (Fujisawa et al., 1990). To elucidate the mechanism of the initiation events relating to packaging specificity, it is necessary to define the structure and the function of sequences necessary for DNA packaging more precisely. We have used the plasmid packaging/transduction system to determine the minimum sequences necessary for DNA packaging. Recently, Chung and Hinkle (1990a,b) described a similar plasmid packaging system for T7. They confirmed the bipartite structure of DNA sequences necessary for packaging T7 DNA and the presence of an RNA polymerase promoter associated with DNA replication in pacB. From the analysis of the effects of altering the spacing and orientation of the two sequences, they suggested an intimate relationship between transcription and packaging. In this paper, the minimum DNA sequences necessary for packaging of T3 DNA were determined by monitoring transduction of deletion mutants of a recombinant plasmid, pUCEl-TR. We will present evidence that transcription is necessary but not sufficient for packaging. The right-half sequence of the pacC (pacCR), target sequence for initiation cleavage, is essential for efficient DNA packaging but the left one (pacCL) is not required. We also present evidence that T3 DNA packaging machinery has an ability to terminate packaging by a headful, nonspecific mechanism in the absence of pacCL. MATERIALS Bacteria,
AND METHODS
phages, and plasmids
Escherichia co/i NG30 (su-, recA-) was used for propagation of T3 phage. NG30 was used as a recipient for plasmid transduction. T3 mutants were from our laboratory stocks and the following mutants were used (the gene numbers are indicated in parentheses): amNG20 (3) amNG64 (4) amNG69 (5), and amNG64 (6). All of these phage strains carried the lysis gene mutation, amNG220. T4 dc mutant (42- 56- denB unf) was described in a previous paper (Fujisawa et al., 1985). Plasmids pUCTR, pUCE1, and pUCEl-TR are pUCl8 plasmids carrying the terminally redundant sequence and its flanking sequences from T3 concatemerit DNA (TR), the T3 genetic right end sequence including gene 19 (El) and a combination of both (El -
TR), respectively (Hashimoto and Fujisawa 1988; Fig. 1, lane 1). CsCl equilibrium density gradient centrifugation was performed as described in a previous paper (Nakasu et a/., 1983). Purification packaging
of phage proteins
involved
in DNA
Proheads were purified according to Nakasu et al. (1985). Gp18 and gpl9 were purified as described in a previous paper (Hamada et a/., 1986a). Buffers and enzymes Complete pat buffer was 5% (w/v) polyethylene glycol (6000), 50 ,uLMATP in 20 mMTris-HCI (pH 7.4) 50 mM NaCI, 1 mM spermidine-5 mM MgCI,, 7 mM 2mercaptoethanol. Prohead buffer is described in a previous paper (Nakasu et al,, 1983). Restriction enzymes and exonuclease III were obtained from Takara Company and used as described by the supplier. In vitro DNA packaging
reaction
A standard reaction mixture (20 ~1) contained 10” phage equivalents (peq) of mature T3 DNA or 0.5 yg DNA, 2 X 10” peq of proheads, 20 pmol of gp18, and 3 pmol of gpl9 in complete pat buffer (Shibata et al., 1987). Assay for plasmid transduction Plasmid transduction was performed as described in a previous paper (Hashimoto and Fujisawa, 1988). Briefly, a 0.2-ml aliquot of recipient strain NG30 was added with a sample containing transducers (20 ~1) and, after incubation for 10 min at 30” for adsorption, the cells were plated with a 2-ml top agar on a membrane filter disk placed on an LB plate. After incubation for 2 hr at 37” to express the resistance marker (Amp?, the filter discs were transferred to LB agar plates containing ampicillin (25 yglml) and incubated for 24 hr at 37”. Assay for the promoter
activity
NG30 cells were infected with T3 3-1~s at a multiplicity of 7 at 30”. At 9 mm after infection, cells were sedimented, washed with buffer ITris-HCI (pH 8.0) 7 mM 2-mercaptoethanol] and resuspended in the same buffer. The cells were disrupted by sonication and the sonicate was centrifuged. The supernatant was used as a preparation of T3 RNA polymerase after the addition of rifampicin (100 pg/ml). Transcription reactions were carried out in a volume of 100 ~1 containing 20 mM Tris-HCI (pH 8.0); 10 mM MgCI,; 7 mM 2-mercaptoethanol; 2 mM spermidine; 0.4 mM GTP, ATP,
790
HASHIMOTO
and CTP; 0.1 mM [a32P]-CTP; 2 pug plasmid DNA; and 20 ~1 of cell extract containing T3 RNA polymerase. After 15 min of synthesis at 37”, radioactivity incorporated into acid-insoluble material was counted. The promoter activity of plasmid DNA was given as percentage of acid-insoluble radioactivity arising from the promoter in pUCAfAI-TR.
Preparation of intracellular DNA Intracellular DNA was prepared according to the method described by Rabkin and Richardson (1988). Briefly, cells were grown to a density of 5 x 1O* cells/ml and infected with T3 at a multiplicity of 7. Cells (5 ml) were collected at 5 and 20 min after infection and suspended in 0.1 vol of ice-cold lysis buffer, 50 mM TrisHCI (pH 7.4), 2 mM EDTA, 10% sucrose. After treatment with lysozyme, the mixture was incubated with pronase K at 55” at 15 min. Cellular DNA and RNA were precipitated by the addition of NaCl to 1 M and an overnight incubation at 4”. After centrifugation, the supernatant was extracted with phenol/chloroform/isoamylalcohol. T3 and plasmid DNAs were precipitated with 125 mM ammonium acetate and ethanol, collected by centrifugation, and dissolved in 10 mM TrisHCI (pH 8.0) 1 mM EDTA.
Agarose gel electrophoresis After the addition of l/5 vol of sample buffer (20% sucrose, 100 mM EDTA, 0.05% bromophenol blue, 1% SDS), the samples were heated at 60” for 10 min and subjected to 0.3-l O/Oagarose gel as described in a previous paper (Shibata et al., 1987).
Determination of the ends of packaged DNA The packaged DNA from pUCAfAI-CR or -CL was prepared from particles purified through a CsCl equilibrium density gradient as described in a previous paper (Hashimoto and Fujisawa, 1988). The full-length transducing DNAs were used as templates after being denatured by heating. The left or right end was determined by measuring the length from 32P-labeled Ml 3 primer, MV or M4, to one end of the DNA, respectively. Sanger’s chain termination method (Sanger el al., 1977) was used from a corresponding 32P-labeled primer directly on the plasmid pUCAfAI-CR or -CL DNA (Chen and Seeburg, 1985).
RESULTS DNA sequences necessary for packaging of T3 DNA In a previous paper (Hashimoto and Fujisawa, 1988) we showed that T3 phage can package and transduce a recombinant plasmid, pUCE1 -TR, carrying a genetic
AND FUJISAWA
right-end 2.7-kb sequence (El) and a concatemerjoint sequence (TR) (Fig. 1, lane 1). A recombinant plasmid carrying the TR sequence alone produced few, if any, transducing particles in spite of the presence of a target sequence for concatemer processing (Fig. 1, lane 2, pUCTR). We tried to determine the minimal functional sequence (pat) necessary for packaging of T3 DNA by using this plasmid transducing system. A series of deletions were constructed to narrow down the DNA sequences in the El sequence (pacB) required for DNA packaging. pUCE1 -TR was digested with Pstl and Xbal in the multicloning site and an approximately 1900-bp-long fragment was deleted rightward to +944 from Xbal site by exonuclease III (pUC EdE3-TR) without any effect on the efficiency of transduction (Fig. 1, lane 4). Although cells carrying pUCElTR grew slowly and frequently lost the plasmid during growth in broth with ampicillin, the pUC EdE3-TR plasmid was rather stable. Next, pUCEdAf-TR or PUCEdAc-TR was constructed by self-ligation after removal of 807 bp (+1600 to +794) or 2019 bp (+2700 to +682) fragments from the El sequence in pUCEl-TR by Aflll or Accl digestion, respectively. pUCEdAf-TR was transduced as well as pUCEl-TR but the transduction efficiency of pUCEdAc-TR decreased to that of pUCTR (Fig. 1, lanes 6 and 7), indicating that the 1 13bp sequence between +794 and +682 is responsible for packaging in the El sequence. Interestingly, a promoter sequence for T3 RNA polymerase is found in the 113-bp sequence as was the case for T7 (Chung and Hinkle, 1990). The 113-bp fragment was divided by Alul into a 56-bp (+794 to +739) fragment containing the promoter and a 57-bp (+738 to +682) fragment. Each fragment was inserted into the HindIll-EcoRI site (+429) of pUCTR in the original direction. pUCAfAI-TR carrying the 56-bp Aflll-Alul fragment was transduced as efficiently as pUCEdE3-TR (Fig. 1, lane 8) but pUCAIAc-TR carrying the 57-bp Alul-Accl fragment was packaged at the same low efficiency as was pUCTR (data not shown). These results suggest that the T3 RNA polymerase promoter sequence is an essential element in the pacB sequence. The TR sequence contains the target for processing of concatemer DNA (pacC), which is composed of the initiation cleavage site at the right end of the terminally redundant sequence and the termination cleavage site at the left end of the terminally redundant sequence that is cleaved to separate monomer DNA from the concatemer. To minimize pacC, the left (pacCL) or right half (pacCR) of the concatemerjoint was removed by using /-/pal site (+140) in the terminally redundant sequence and flanking fcoRl sites (+429 and -86). A deletion removing the left half of the concatemer joint
DNA SEQUENCES
NECESSARY
FOR PACKAGING
BACTERIOPHAGE
Multi cloning site 1 1
1
pUCElTR
2
pU(J’R
3
pUCE1
4
pUCEdE3 TR
5
pUCDK7
6
PUCAf TR
7
pUCAc TR
8
puc.kfid TR
9
pUCAfAl CL
lo
pUCAfAl CR
791
13 DNA
transduction Crrd/ml)
“I
I
lsu
lYsFss3
I
cl
I
Cl
rxm
1
I
-
11 pUClg FIG. 1. A schematic presentation of T3 DNA fragments cloned into pUCl8. The numbers are the nucleotides numbered leftward or rightward (-) from the genetic right end. Hatched and open boxes indicate the terminally redundant and its flanking sequences from concatemer DNA, respectively. Restriction enzyme sites are indicated: Act, Accl; Afl, Aflll; Alu. Alul; Eco, EcoRI; Hind, HindIll; Hpa, /-/pal; Pst, Pstl; Xba, Xbal. The transducing activity was assayed as described under Materials and Methods and represents an average of transducers per milliliter obtained in two to three separate experiments.
(pUCAfAI-CR) was packaged as efficiently as pUCAfAITR (Fig. 1, lanes 8 and lo), but a deletion removing the right end (pUCAfAI-CL) was packaged at a very low efficiency as was pUCE1 (Fig. 1, lanes 3 and 9). These results indicate that the left end of the concatemerjoint (pacCL) is not required for efficient packaging by T3 phage. A series of deletions of the CR sequence in pUCAfAI-CR was constructed to define more precisely the minimal pacCR sequence. Deletions to pUCAfAICR-l 1 R, which retained 11 bp to the right end of the terminally redundant sequence from the right, had full packaging activity (Fig. 2, lane 5) but a deletion to pUCAfAI-CR-3, which retained 3 bp to the right end, showed a significant drop in the efficiency of transduction in spite of the intact right end cleavage site (Fig. 2, lane 4). pUCAfAI-CR could be deleted to 10 bp to the
right end from the left (pUCAfAI-CR+lOL) without effect on the production of transducers (Fig. 2, lane 1). Analysis of the ends of the packaged and pUCAfAI-CL DNAs
pUCAfAI-CR
As shown above, pUCAfAI-CR, lacking ,oacCL, was packaged as efficiently as pUCEdE3-TR but pUCAfAICL, lacking pacCR, was packaged at a very low efficiency (Fig. 1, lanes 9 and 10). To get insight into the mechanism of packaging of these plasmid DNAs, DNA in transducing particles was analyzed. In a previous paper (Hashimoto and Fujisawa, 1988), we showed that DNA from pUCEI-TR transducing particles are head-to-tail oligomers (mainly hexamer) of plasmid DNA with the same termini as those of mature T3 DNA.
792
HASHIMOTO 1
Hpal
-11
-3
Dependence
t
+ ,0&G= TCTFTTAGCCCCTTA .
Trd/ml
d/,/
1 CR*IOL
2 CR-4L 3
CR+lR
6.8X10' +-I,-
-,,;b
8.0X104 Z.ZXI05
4 CR-3R
9.0x10’
5 CR-11R
4.0x1
z-
AND FUJISAWA
0’
FIG. 2. A schematic presentation of deleted derivatives of the pacCR sequence derived from pUCAfAI-CR. Numbering is the same as described in the legend to Fig. 1. A box and an underline represent the terminally redundant and its flanking sequences in the pacCR, respectively. The horizontal arrows indicate remaining DNA in deletion derivatives of thepacCR sequence. The transducing activity was assayed and is represented as described in the legend to Fig. 1.
Phages and transducing particles were subjected to CsCl equilibrium density gradient centrifugation and the fractions from the gradient were analyzed for amp’ transformants. As shown in Fig. 3A, amp’ transductants from pUAfAI-CR yielded a single peak, banding at a slightly higher density than the phage particles, although in a broader distribution. On the other hand, amp’ transformants from pUCAfAI-CL yielded two peaks, banding at slightly higher (fractions 6, 7) and lower (fractions 10, 11) densities than phage particles (Fig. 3B). To determine the ends of packaged DNA, DNA was prepared from transducing particles purified through CsCl equilibrium density gradient. The left or right end was determined by primer extension using an appropriate 32P-labeled primer as described under Materials and Methods. With pUCAfAI-CR (Fig. 4A), the sequence toward the right ended at 5’-TGTGTCCCT-3’, corresponding to the right-end sequence of mature T3 DNA (see Fig. 2) but no unique band corresponding to the left end was detected (data not shown). With pUCAfAI-CL (Fig. 4B), the sequence toward the left ended at 5’-TCTCATAGTT-3’, corresponding to the left end of mature T3 DNA (Fujisawa and Sugimoto 1983) but no unique band corresponding to the right end was detected (data not shown). Judging from the density distribution, the transducing pUCAfAI-CR and pUCAfAICL particles appear to contain plasmid oligomers with sizes of headful length (about 40 kb; Hashimoto and Fujisawa, 1988). These results indicate that DNA in pUCAfAI-CR or pUCAfAI-CL transducing particles is plasmid oligomer and has the same right or left end as that of mature T3 DNA and a heterologous, nonunique left or right end, respectively.
of transduction
on promoter
activity
As shown above, a region containing a T3 RNA polymerase promoter, located at 98.0% genomic position, was essential for efficient transduction of recombinant plasmids. To analyze the relationship between the promoter and the DNA packaging activities more directly, a series of deletion mutations were constructed that extend into the 56-bp Aflll-Alul region in pUCAfAI-TR (containing a promoter for T3 RNA polymerase) from either the right or the left, as follows. An AfAl fragment from pUCAfAl CR, in which the fragment was flanked by HindIll and Pstl sequences, was cloned between HindIll and Pstl sites of pUC18 (pUCAfAI). The AfAl sequence was deleted rightward or leftward by mild exonuclease III digestion from the /-findIll site after Hindill digestion or from the BamHl site after BamHIKpnl double digestion of pUC AfAl and circularized after adding HindIll or Kpnl linkers, respectively. Deletions were confirmed by DNA sequencing. The TR fragment was inserted into appropriate deleted plasmids and recombinant plasmids carrying the TR se-
I
1
I
I
I
I
5
10
15
5
10
15
Fraction
J
No.
FIG. 3. Density gradient centrifugation of pUCAfAI-CR and pUCAfAI-CL transducing particles. NG30 cells carrying pUCAfAI-CR (A) or pUCAfAI-CL (B) were infected with T3 33. After incubation for 60 min, cells were collected and lysed with chloroform. Transducing particles were concentrated by two cycles of low- and high-speed centrifugation and subjected to CsCl equilibrium centrifugation and plaque-forming units on Rl 1S (su+) (0) or amp’transducers (0) were measured with all fractions as described under Materials and Methods. Fractions are left to right corresponding to from bottom to top.
DNA SEQUENCES
NECESSARY
FOR PACKAGING
ACGTN
ACGTN
BACTERIOPHAGE
793
T3 DNA
sensus sequences for T3 RNA polymerase (R + 7, +3, and +l) while the promoter activity was not impaired until deletion of G (+l) at the start site of transcription (R + 3, +l, and -6). These results indicate that the promoter activity in the pacB sequence is necessary but not sufficient for DNA packaging.
B
DISCUSSION
/
In a previous paper (Hashimoto and Fujisawa, 1988) we showed that a recombinant DNA carrying El and TR sequences from T3 concatemer DNA can efficiently be packaged into transducing particles. We used this in viva plasmid transduction as an assay system to define the minimal sequences necessary for DNA packaging (pat sequence). The TR sequence contains the target sequences for the initiation and termination cleavages, pacCR and CL, respectively. Unexpectedly, deletion experiments demonstrated that pacCR was essential but pacCL was unnecessary for efficient packaging of ,plasmid DNA. pUCAfAI-CR transducing
7 FIG. 4. The end of DNAfrom pUCAfAI-CR or pUCAfAI-CL transducing particles. Sanger’s chain termination method was used. from Ml 3 primer M4 or MV directly on circular pUCAfAI-CR (A) or pUCAfAI-CL (B), respectively. The sequence of the template is shown. The region near expected length of the mature end is shown because no other prominent bands were seen. Lane N shows the position of the end of the template DNA. (A) The sequence toward the right-end of pUCAfAI-CR was determined using 32P-labeled Ml3 primer (M4) which anneals to pUCl8 sequence left of the HindIll site. Numbering is the same as described in the legend to Fig. 1. (B) The sequence toward the left-end of pUCAfAI-CL was determined using 32P-labeled Ml3 primer (MV) which anneals to pUC18 sequence right of the EcoRl site. The nucleotides are numbered leftward (-) or rightward from the genetic left end.
quence in the same direction as in pUCAfAI TR were selected for further analysis. The promoter and packaging activities were determined by assaying for in vitro transcription and in vivo transduction, respectively, as described under Materials and Methods (Fig. 5). For deletions from the left, the efficiency of transduction began to drop as deletions entered into the consensus sequence for T3 RNA polymerase (-17 to +6) in parallel with a decrease in the promoter activity (L-l 1, L-9). For deletions from the right, the efficiency of transduction began to drop as deletions entered into the con-
L-28 L-23
4 4
L-11 L-9
4
R*l R-6
* *
FIG. 5. Relationship of the transducing efficiency to the promoter activity. The AfAl sequence of pUCAfAI-TR was deleted by exonuclease Ill from the left (L series) or the right (R series). The nucleotides are numbered leftward (-) or rightward (+) from the first nucleotide transcribed by T3 RNA polymerase. The consensus promoter sequence for T3 RNA polymerase extends from -17 to +6. An underline indicates a specific binding promoter sequence of T3 RNA polymerase. Horizontal arrows indicate deletion derivatives of the AfAl fragment. The promoter activity (0) and the transducing activity (0) were assayed as described under Materials and Methods.
794
HASHIMOTO
particles contained plasmid oligomers with the same right end as T3 and a nonunique left end. These results indicate that T3 DNA packaging machinery requires the specific sequence, pacCR, to create the DNA end essential for initiation of packaging and that the second termination cleavage can operate by a headful mechanism at the specific sequence in pacCL or at any sequence if pacCL is deleted. In the absence of pacCR, the packaging machinery would produce an end for packaging by a nonspecific cleavage at a low efficiency and cleave the pacCL sequence at the site used to form the left end of mature T3 DNA after packaging a headful of DNA (T3 DNA, 38.74 kb) (Bailey eta/., 1980), corresponding to sizes of 12 or 13 plasmid monomers (3 kb), resulting in the production of two peaks (Fig. 2B), as observed for pUCEl-TR (Hashimoto and Fujisawa, 1988). For T7 phage, Chung and Hinkle (1990b) report plasmids which contain either the pacCL or CR are packaged at about 10% of the efficiency of those with both sequences. The residual packaging of T7 plasmids results from regeneration of pacCL or CR by recombination with replicating T7 DNA or recognizing a site in pBR322 (Chung and Hinkle, 1990a,b). For T3 phage, the indispensability of pacCR appears to be unmasked because T3 3-, which was used as a helper phage in the experiment, was defective in recombination. Our experiments demonstrate that pacCR can be defined to be a 21 -bp region (R + 10 to R - 1 1) flanking the cleavage site, generating the right end. A short sequence CCTAAAG or its variants, which are repeated many times on each site of and within the terminally redundant sequence in both T3 and T7 (Dunn and Studier, 1983; Yamada et al. 1986), can be deleted without affecting the efficiency of plasmid packaging by both T3 (this paper) and T7 (Chung and Hinkle, 1990b). In a previous paper (Fujisawa et al., 1990) we showed that the cleavage at the left end of the terminally redundant sequence in pacCL did not require ,oacCR in the defined in vitro system. In in viva packaging/transduction system, the cleavage at the right end of the terminally redundant sequence in pacCR occurred efficiently in the absence ofpacCL (Fig. 1, lanes 5 and 10). These results argue against a model for DNA processing of T3IT7 phage in which DNA maturation requires both ends of the terminally redundant sequence in pacC (Watson, 1972). We have defined the functional element in the El sequence to be a 34-bp sequence, corresponding to the consensus promoter sequence protected by T3 RNA polymerase (-21 to +l 1) in the presence of GTP or GTP + ATP (Basu and Maitra, 1986). Deletions of the upstream sequence from the left decreased both the promoter and the transducing activities in parallel
AND FUJISAWA
(Fig. 5). On the other hand, deletions of the downstream sequences from the right impaired the transducing activity before the promoter activity (Fig. 5). These results indicate that the transcriptional activity is necessary but not sufficient for DNA packaging. The conclusion is consistent with the observation that transcription is required but the T7 promoter alone is not active for plasmid transduction by T7 (Chung and Hinkle, 1990b). A small viral (Guo et a/., 1987) or nonviral (Donate and Carrascosa, 1991) RNA is required for DNA packaging by $29 or X phage, respectively. However, DNA packaging of T3 phage is not inhibited but rather stimulated by RNase A treatment both in the defined or crude in vitro DNA packaging system. It seems likely that, instead of a specific RNA transcript, it is the act of transcription itself that is required for concatemer processing and packaging of T3 DNA. Although T3 RNA polymerase initiates transcription depending on a sequence -21 to +1 G (Fig. 5, R + l), the packaging requires at least transcription of a following GA stretch (+2A to +I 2G). A strong T3 promoter, 410, lacking a GA stretch, did not work as pacB (data not shown). The binding of T3 RNA polymerase becomes more stable when the GA stretch is transcribed in the presence of both ATP and GTP (Basu and Maitra, 1986). The packaging machinery may land on the pat signal with a directionality through interaction with a stable T3 RNA polymerase-promoter complex. A direct interaction between RNA polymerase and a packaging protein, gpl9, is suggested for T7 (Studier, personal communication). Although DNA negative mutations of T3 are not deficient in morphogenetic proteins (Fujisawa eta/., 1978) transduction requires gene products involved in DNA replication (Hashimoto and Fujisawa, 1988). However, plasmid DNA did not amplify replication of a short specific sequence(s). With T7, only the promoters associated with DNA replication are effective in DNA packaging (Chung and Hinkle, 1990b) and DNA synthesis is required for the production of the left-hand terminally redundant sequence during DNA packaging (White and Richardson, 1987). The relationship of transcription and DNA replication to DNA packaging remains to be elucidated. ACKNOWLEDGMENTS We are grateful to Dr. Michael Feiss (The University of Iowa) for his invaluable help with the manuscript. This work is supported by the Mitsubishi Foundation and a grant-in-aid for scientific research from the Ministry of Education of Japan.
REFERENCES BAILEY, J. N., DEMBINSKI, D. R., and MCALLISTER,
W. T. (1980). Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA. J. Viral. 35, 176-l 83.
DNA SEQUENCES
NECESSARY
FOR PACKAGING
BEHNISH,W., and SCHMIEGER,H. (1985). ln v&o packaging of plasmid DNA oligomers by Saimoneiia phage P22 independent of the pat site, and evidence for the termination cut in virro. Virology 144, 310-317. BLACK, L. W. (1981). The mechanism of bacteriophage DNA encapsidation. f’rog. C/in. Biol. Res. 64, 97-1 10. BLJKHARI,A. Z., FROSHAUER,S., and BOTCHAM, M. (1976). Ends of bacteriophage Mu DNA. Mafure (London) 264, 580-583. BASU, S:, and MAITRA, U. (1986). Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleotide triphosphate (GTP). i. Mol. Bioi. 190, 425-437. CASJENS,S., and HUANG, W. M. (1982). Initiation of sequential packaging of bacteriophage P22 DNA. J. Mol. Biol. 157, 287-298. CHEN, E. Y., and SEEEURG,P. H. (1985). Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4, 165170. CHUNG, Y. B., and HINKLE, D. C. (1990a). Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection. J. Mol. Biol. 216, 911-926. CHUNG, Y. B., and HINKLE, D. C. (1990b). Bacteriophage T7 DNA packaging. II. Analysis of the DNA sequences required for packaging using a plasmid transducing assay. J. Mol. Biol. 216,927-938. DONATE, L. E., and CARRASCOSA,J. L. (1991). Characterization of versatile in vitro DNA-packaging system based on hybrid lambda/phi29 proheads. Virology 182, 534-544. DUNN, J. J., and STUDIER,F. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535. EARNSHAW,W. C.. and CASJENS,S. R. (1980). DNA Packaging by the double-stranded DNA bacteriophages. Cell 21, 319-331. FEISS, M., KOBAYASHI, I., and WIDNER, W. (1983). Separate sites for binding and nicking of bacteriophage X DNA by terminase. Proc. Nat/ Acad. Sci USA 80, 955-959. FUJISAWA,H., KIMURA, M., and HASHIMOTO, C. (1990). In vifro cleavage of the concatemer joint of bacteriophage T3. \/iro/ogy 174, 26-34. FUJISAWA,H., MIYAZAKI, J., and MINAGAWA, T. (1978). in v&-o packaging of phage T3 DNA. Virology 87, 394-400. FUJISAWA,H., and SUGIMOTO, K. (1983). On the terminally redundant sequences of bacteriophage T3 DNA. Virology 124, 251-258. FUJISAWA,H., and YAMAGISHI, M. (1981). Studies on factors involved in in vitro packaging of phage T3 DNA. Prog. C/in. Biot. Res. 64, 239-252. FUJISAWA,H., YONESAKI, T., and MINAGAWA, T. (1985). Sequence of the T4 recombination gene, UVSX, and its comparison with that of the recA gene of Escherichia coli. Nucl. Acids Res. 13, 74737481. GEORGE, M., and BUKHARI, A. (1981). Heterogenous host DNA attached to the left end of mature bacteriophage Mu DNA. Nature (London) 292, 175-l 76. Guo, P., ERICKSON,S., and ANDERSON, D. L. (1987). A small viral RNA is required for in v&o packaging of bacteriophage phi-29. Science 236, 690-694.
BACTERIOPHAGE
T3 DNA
795
HAMADA, K., FUJISAWA,H., and MINAGAWA, T. (1986a). Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging. Virology 151,ll O118. HAMADA, K., FUJISAWA,H., and MINAGAWA, T. (1986b). A defined in vitro system for packaging of bacteriophage T3 DNA. Virology 151,119-123. HASHIMOTO, C., and FUJISAWA,H. (1988). Packaging and transduction of non-T3 DNA by bacteriophage T3. virology 166,432-439. KUT~ER, E., GUI-~MAN, B., MOSIG, G., and RUEGER,W. (1990). Genomic map of bacteriophage. In “Genetic Maps” (S. J. O’Brien, Ed.), pp. 1.24-l .51. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MURIALDO, H.. and BECKER,A. (1978). Head morphogenesis of complex double-stranded DNA bacteriophage. Microbial. Rev. 42, 529-576. NAKASU, S., FUJISAWA,H., and MINAGAWA, T. (1983). Role of gene 8 product in morphogenesis of bacteriophage T3. L/irology 127, 124-133. RABKIN,S. D., and RICHARDSON,C. C. (1988). Initiation of DNA replication at cloned origins of bacteriophage T7. J. Mol. B/o/. 204, 903916. RAMSEY, N., and RITTCHIE, D. A. (1983). Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophageT1 DNA packaging. Nature (London) 264, 264-266. ROBERTS,S., SHELDON, R., and SADOWSKI, P. D. (1978). Genetic recombination of bacteriophage T7 in vitro. IV. Asymmetry of recombination frequencies caused by polarity of DNA packaging. tirology 89, 252-261. SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat/. Acad. Sci. USA . 74, 5463-5467. SHIBATA, M., FUJISAWA,H., and MINAGAWA,T. (1987). Early events in a defined in vitro system for packaging of bacteriophage T3 DNA. Virology 159, 250-258. STERNBERG,N., and COULBY, J. (1987). Recognition and cleavage of the bacteriophage Pl packaging site(pac). Il. Functional limits of pat and location of pat cleavage termini. 1. Mol. Biol. 194, 469479. STREISINGER,G., EMRICH, J., and STAHL, M. M. (1967). Chromosome structure in phageT4. Ill. Terminal redundancy and length determination. Proc. Natl. Acad. Sci. USA. 57, 292-295. WATSON, J. D. (1972). Origin of concatemeric T7 DNA. Nature New Biol. 239, 197-201. WHITE, J. H., and RICHARDSON, C. C. (1987). Processing of concatemers of bacteriophage T7 DNA in vitro. J. Biol. Chem. 262, 8851-8860. Wu, R., and TAYLOR, E. (1971). Nucleotide sequence analysis of DNA. Il. Complete nucleotide sequence of the cohesive ends of bacteriophage X DNA. J. Mol. Biol. 57, 491-51 1. YAMADA, M., FUJISAWA,H., KATO, H., HAMADA, K., and MINAGAWA, T. (1986). Cloning and sequencing of the genetic right end of bacteriophage T3 DNA. Virology 151, 350-361. YAMAGISHI, M., FUJISAWA,H., and MINAGAWA, T. (1985). Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA packaging specificity. Virology 144, 502-515.
i
87,788-795
(1992)
DNA Sequences
Necessary
for Packaging
CHIKARA HASHIMOTO Department
AND
Bacteriophage
HISAO FUJISAWA’
of Botany, Faculty of Science, Kyoto University,
Received October
T3 DNA
16, 199 1; accepted
December
Kyoto 606, Japan 27, 199 1
A recombinant plasmid, pUCEl-TR, carrying a target for processing of the concatemer joint (TR) and sequences to the left of the target (El), is efficiently packaged into transducing particles during T3 phage infection. Using this plasmid packaging/transduction system, the minimal sequences necessary for packaging of T3 DNA were determined. The TR sequence contains the targets for initiation cleavage and termination cleavage of concatemer processing (pacCR and pacCL, respectively). A plasmid lacking pacCL was packaged as efficiently as pUCE1 -TR but one deleted forpacCR was packaged at a very low efficiency, showing that pacCR is essential for production of transducers but that pacCL is dispensable. DNA from transducing particles carrying a recombinant plasmid lacking pacCL or pacCR had the same right or left end as T3 DNA, respectively, but its other end was not unique. In the absence of pacCL, packaging is initiated from the DNA end created by cleavage at thepacCR and terminated at any sequence after packaging a headful of DNA. In the absence of pacCR, packaging is initiated from the DNA end created by nonspecific, inefficient cleavage and terminated by cleavage at the pacCL after packaging a headful of DNA. A 23-bp segment flanking the site where the mature right end is formed was found to support efficient formation of transducing particles. A 53-bp sequence, including a consensus sequence for the promoter for T3 RNA polymerase, was a responsible element in the El sequence for packaging of plasmid DNA. Deletions of the 5’-upstream sequence of the promoter sequence from the left decreased the promoter and packaging activities in parallel, but with those of the 3’-downstream sequence from the right, the packaging activity was impaired before the promoter activity, indicating that transcription from the promoter is necessary but not sufficient for T3 DNA packaging. 0 1992 Academic Press, Inc.
INTRODUCTION
ture monomers are cut from the concatemer. In a previous paper (Hashimoto and Fujisawa, 1988), we showed that a recombinant plasmid carrying the concatemer joint (TR) sequence, including the terminally redundant sequence, and its left side flanking (El) sequences from T3 concatemeric DNA (pUCE1 -TR; Fig. 1, lane I), can be packaged into transducing particles. Transducing T3 particles contained head-to-tail oligomers of plasmid DNA with the same termini as those of mature T3 DNA. Thus, the T3 concatemer joint sequence in the plasmid is recognized for processing and packaging by T3 phage. However, the TR sequence alone, containing the cleavage sites for processing of concatemeric DNA (pacC), is not sufficient and the El sequence is additionally required for recognition by the packaging machinery (tentatively named pacB). From these results, we proposed that a bipartite structure of the sequence necessary for DNA packaging (pat signal), consisting of pacB and pacC, may be general for the sequences required for initiation cleavage in DNA packaging [Mu (George and Bukhari, 1981) P22 (Casjens and Huang, 1982), X (Feiss, et a/., 1982), and Pl (Sternberg and Coulby, 1987)]. In a defined in vitro system for packaging T3 DNA which is composed of purified proheads and packaging proteins (Fujisawa et al., 1990), plasmid DNAs containing T3 concatemer joints are cleaved at the left end of the terminally redundant sequence after being packaged leftward (Fujisawa eT
During the growth of most double-stranded DNA bacteriophages, phage DNA is synthesized as concatemers and packaged into head precursors, called proheads, as mature monomers are cut from the concatemer. The cutting of concatemeric DNA in viva is coupled to headful packaging (see reviews: Murialdo and Becker, 1978; Earnshaw and Casjens, 1980; Black, 1981). Cutting can be strictly sequence specific as in X (Wu and Taylor, 197 1), T3 (Fujisawa and Sugimoto, 1983), and T7 (Dunn and Studier, 1983). In the case of Mu (Bukhari et al, 1976), P22 (Casjens and Huang, 1982) Tl (Ramsey and Rittchie, 1983) or Pl (Sternberg and Coulby, 1987) cutting is initiated from a specific packaging sequence called pat and terminated at any sequence after packaging a headful of DNA. In the case of T4, heads can be filled without any apparent sequence specificity by a headful mechanism (Streisinger et a/, 1967). The termini of mature DNA from phage T3 contain the same 230 base pair sequences, called terminally redundant sequences. DNA is synthesized as concatemers in which unit-length molecules are joined together in a head-to-tail fashion through the terminally redundant sequences. During packaging of DNA, ma’ To whom reprint requests should be addressed. 0042.6822/92
$3.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
788
789
DNA SEQUENCES NECESSARY FOR PACKAGING BACTERIOPHAGE T3 DNA
a/., 1990). Therefore, the cleavage reaction in the in vitro system would correspond to the termination cleavage in vivo. Although T3 and related phage T7 DNAs can be discriminated during packaging, the termination cleavage is not specific between T3 and T7. From these results, we conclude that the packaging specificity resides in the initiation event(s), including the initiation cleavage, during DNA packaging (Fujisawa et al., 1990). To elucidate the mechanism of the initiation events relating to packaging specificity, it is necessary to define the structure and the function of sequences necessary for DNA packaging more precisely. We have used the plasmid packaging/transduction system to determine the minimum sequences necessary for DNA packaging. Recently, Chung and Hinkle (1990a,b) described a similar plasmid packaging system for T7. They confirmed the bipartite structure of DNA sequences necessary for packaging T7 DNA and the presence of an RNA polymerase promoter associated with DNA replication in pacB. From the analysis of the effects of altering the spacing and orientation of the two sequences, they suggested an intimate relationship between transcription and packaging. In this paper, the minimum DNA sequences necessary for packaging of T3 DNA were determined by monitoring transduction of deletion mutants of a recombinant plasmid, pUCEl-TR. We will present evidence that transcription is necessary but not sufficient for packaging. The right-half sequence of the pacC (pacCR), target sequence for initiation cleavage, is essential for efficient DNA packaging but the left one (pacCL) is not required. We also present evidence that T3 DNA packaging machinery has an ability to terminate packaging by a headful, nonspecific mechanism in the absence of pacCL. MATERIALS Bacteria,
AND METHODS
phages, and plasmids
Escherichia co/i NG30 (su-, recA-) was used for propagation of T3 phage. NG30 was used as a recipient for plasmid transduction. T3 mutants were from our laboratory stocks and the following mutants were used (the gene numbers are indicated in parentheses): amNG20 (3) amNG64 (4) amNG69 (5), and amNG64 (6). All of these phage strains carried the lysis gene mutation, amNG220. T4 dc mutant (42- 56- denB unf) was described in a previous paper (Fujisawa et al., 1985). Plasmids pUCTR, pUCE1, and pUCEl-TR are pUCl8 plasmids carrying the terminally redundant sequence and its flanking sequences from T3 concatemerit DNA (TR), the T3 genetic right end sequence including gene 19 (El) and a combination of both (El -
TR), respectively (Hashimoto and Fujisawa 1988; Fig. 1, lane 1). CsCl equilibrium density gradient centrifugation was performed as described in a previous paper (Nakasu et a/., 1983). Purification packaging
of phage proteins
involved
in DNA
Proheads were purified according to Nakasu et al. (1985). Gp18 and gpl9 were purified as described in a previous paper (Hamada et a/., 1986a). Buffers and enzymes Complete pat buffer was 5% (w/v) polyethylene glycol (6000), 50 ,uLMATP in 20 mMTris-HCI (pH 7.4) 50 mM NaCI, 1 mM spermidine-5 mM MgCI,, 7 mM 2mercaptoethanol. Prohead buffer is described in a previous paper (Nakasu et al,, 1983). Restriction enzymes and exonuclease III were obtained from Takara Company and used as described by the supplier. In vitro DNA packaging
reaction
A standard reaction mixture (20 ~1) contained 10” phage equivalents (peq) of mature T3 DNA or 0.5 yg DNA, 2 X 10” peq of proheads, 20 pmol of gp18, and 3 pmol of gpl9 in complete pat buffer (Shibata et al., 1987). Assay for plasmid transduction Plasmid transduction was performed as described in a previous paper (Hashimoto and Fujisawa, 1988). Briefly, a 0.2-ml aliquot of recipient strain NG30 was added with a sample containing transducers (20 ~1) and, after incubation for 10 min at 30” for adsorption, the cells were plated with a 2-ml top agar on a membrane filter disk placed on an LB plate. After incubation for 2 hr at 37” to express the resistance marker (Amp?, the filter discs were transferred to LB agar plates containing ampicillin (25 yglml) and incubated for 24 hr at 37”. Assay for the promoter
activity
NG30 cells were infected with T3 3-1~s at a multiplicity of 7 at 30”. At 9 mm after infection, cells were sedimented, washed with buffer ITris-HCI (pH 8.0) 7 mM 2-mercaptoethanol] and resuspended in the same buffer. The cells were disrupted by sonication and the sonicate was centrifuged. The supernatant was used as a preparation of T3 RNA polymerase after the addition of rifampicin (100 pg/ml). Transcription reactions were carried out in a volume of 100 ~1 containing 20 mM Tris-HCI (pH 8.0); 10 mM MgCI,; 7 mM 2-mercaptoethanol; 2 mM spermidine; 0.4 mM GTP, ATP,
790
HASHIMOTO
and CTP; 0.1 mM [a32P]-CTP; 2 pug plasmid DNA; and 20 ~1 of cell extract containing T3 RNA polymerase. After 15 min of synthesis at 37”, radioactivity incorporated into acid-insoluble material was counted. The promoter activity of plasmid DNA was given as percentage of acid-insoluble radioactivity arising from the promoter in pUCAfAI-TR.
Preparation of intracellular DNA Intracellular DNA was prepared according to the method described by Rabkin and Richardson (1988). Briefly, cells were grown to a density of 5 x 1O* cells/ml and infected with T3 at a multiplicity of 7. Cells (5 ml) were collected at 5 and 20 min after infection and suspended in 0.1 vol of ice-cold lysis buffer, 50 mM TrisHCI (pH 7.4), 2 mM EDTA, 10% sucrose. After treatment with lysozyme, the mixture was incubated with pronase K at 55” at 15 min. Cellular DNA and RNA were precipitated by the addition of NaCl to 1 M and an overnight incubation at 4”. After centrifugation, the supernatant was extracted with phenol/chloroform/isoamylalcohol. T3 and plasmid DNAs were precipitated with 125 mM ammonium acetate and ethanol, collected by centrifugation, and dissolved in 10 mM TrisHCI (pH 8.0) 1 mM EDTA.
Agarose gel electrophoresis After the addition of l/5 vol of sample buffer (20% sucrose, 100 mM EDTA, 0.05% bromophenol blue, 1% SDS), the samples were heated at 60” for 10 min and subjected to 0.3-l O/Oagarose gel as described in a previous paper (Shibata et al., 1987).
Determination of the ends of packaged DNA The packaged DNA from pUCAfAI-CR or -CL was prepared from particles purified through a CsCl equilibrium density gradient as described in a previous paper (Hashimoto and Fujisawa, 1988). The full-length transducing DNAs were used as templates after being denatured by heating. The left or right end was determined by measuring the length from 32P-labeled Ml 3 primer, MV or M4, to one end of the DNA, respectively. Sanger’s chain termination method (Sanger el al., 1977) was used from a corresponding 32P-labeled primer directly on the plasmid pUCAfAI-CR or -CL DNA (Chen and Seeburg, 1985).
RESULTS DNA sequences necessary for packaging of T3 DNA In a previous paper (Hashimoto and Fujisawa, 1988) we showed that T3 phage can package and transduce a recombinant plasmid, pUCE1 -TR, carrying a genetic
AND FUJISAWA
right-end 2.7-kb sequence (El) and a concatemerjoint sequence (TR) (Fig. 1, lane 1). A recombinant plasmid carrying the TR sequence alone produced few, if any, transducing particles in spite of the presence of a target sequence for concatemer processing (Fig. 1, lane 2, pUCTR). We tried to determine the minimal functional sequence (pat) necessary for packaging of T3 DNA by using this plasmid transducing system. A series of deletions were constructed to narrow down the DNA sequences in the El sequence (pacB) required for DNA packaging. pUCE1 -TR was digested with Pstl and Xbal in the multicloning site and an approximately 1900-bp-long fragment was deleted rightward to +944 from Xbal site by exonuclease III (pUC EdE3-TR) without any effect on the efficiency of transduction (Fig. 1, lane 4). Although cells carrying pUCElTR grew slowly and frequently lost the plasmid during growth in broth with ampicillin, the pUC EdE3-TR plasmid was rather stable. Next, pUCEdAf-TR or PUCEdAc-TR was constructed by self-ligation after removal of 807 bp (+1600 to +794) or 2019 bp (+2700 to +682) fragments from the El sequence in pUCEl-TR by Aflll or Accl digestion, respectively. pUCEdAf-TR was transduced as well as pUCEl-TR but the transduction efficiency of pUCEdAc-TR decreased to that of pUCTR (Fig. 1, lanes 6 and 7), indicating that the 1 13bp sequence between +794 and +682 is responsible for packaging in the El sequence. Interestingly, a promoter sequence for T3 RNA polymerase is found in the 113-bp sequence as was the case for T7 (Chung and Hinkle, 1990). The 113-bp fragment was divided by Alul into a 56-bp (+794 to +739) fragment containing the promoter and a 57-bp (+738 to +682) fragment. Each fragment was inserted into the HindIll-EcoRI site (+429) of pUCTR in the original direction. pUCAfAI-TR carrying the 56-bp Aflll-Alul fragment was transduced as efficiently as pUCEdE3-TR (Fig. 1, lane 8) but pUCAIAc-TR carrying the 57-bp Alul-Accl fragment was packaged at the same low efficiency as was pUCTR (data not shown). These results suggest that the T3 RNA polymerase promoter sequence is an essential element in the pacB sequence. The TR sequence contains the target for processing of concatemer DNA (pacC), which is composed of the initiation cleavage site at the right end of the terminally redundant sequence and the termination cleavage site at the left end of the terminally redundant sequence that is cleaved to separate monomer DNA from the concatemer. To minimize pacC, the left (pacCL) or right half (pacCR) of the concatemerjoint was removed by using /-/pal site (+140) in the terminally redundant sequence and flanking fcoRl sites (+429 and -86). A deletion removing the left half of the concatemer joint
DNA SEQUENCES
NECESSARY
FOR PACKAGING
BACTERIOPHAGE
Multi cloning site 1 1
1
pUCElTR
2
pU(J’R
3
pUCE1
4
pUCEdE3 TR
5
pUCDK7
6
PUCAf TR
7
pUCAc TR
8
puc.kfid TR
9
pUCAfAl CL
lo
pUCAfAl CR
791
13 DNA
transduction Crrd/ml)
“I
I
lsu
lYsFss3
I
cl
I
Cl
rxm
1
I
-
11 pUClg FIG. 1. A schematic presentation of T3 DNA fragments cloned into pUCl8. The numbers are the nucleotides numbered leftward or rightward (-) from the genetic right end. Hatched and open boxes indicate the terminally redundant and its flanking sequences from concatemer DNA, respectively. Restriction enzyme sites are indicated: Act, Accl; Afl, Aflll; Alu. Alul; Eco, EcoRI; Hind, HindIll; Hpa, /-/pal; Pst, Pstl; Xba, Xbal. The transducing activity was assayed as described under Materials and Methods and represents an average of transducers per milliliter obtained in two to three separate experiments.
(pUCAfAI-CR) was packaged as efficiently as pUCAfAITR (Fig. 1, lanes 8 and lo), but a deletion removing the right end (pUCAfAI-CL) was packaged at a very low efficiency as was pUCE1 (Fig. 1, lanes 3 and 9). These results indicate that the left end of the concatemerjoint (pacCL) is not required for efficient packaging by T3 phage. A series of deletions of the CR sequence in pUCAfAI-CR was constructed to define more precisely the minimal pacCR sequence. Deletions to pUCAfAICR-l 1 R, which retained 11 bp to the right end of the terminally redundant sequence from the right, had full packaging activity (Fig. 2, lane 5) but a deletion to pUCAfAI-CR-3, which retained 3 bp to the right end, showed a significant drop in the efficiency of transduction in spite of the intact right end cleavage site (Fig. 2, lane 4). pUCAfAI-CR could be deleted to 10 bp to the
right end from the left (pUCAfAI-CR+lOL) without effect on the production of transducers (Fig. 2, lane 1). Analysis of the ends of the packaged and pUCAfAI-CL DNAs
pUCAfAI-CR
As shown above, pUCAfAI-CR, lacking ,oacCL, was packaged as efficiently as pUCEdE3-TR but pUCAfAICL, lacking pacCR, was packaged at a very low efficiency (Fig. 1, lanes 9 and 10). To get insight into the mechanism of packaging of these plasmid DNAs, DNA in transducing particles was analyzed. In a previous paper (Hashimoto and Fujisawa, 1988), we showed that DNA from pUCEI-TR transducing particles are head-to-tail oligomers (mainly hexamer) of plasmid DNA with the same termini as those of mature T3 DNA.
792
HASHIMOTO 1
Hpal
-11
-3
Dependence
t
+ ,0&G= TCTFTTAGCCCCTTA .
Trd/ml
d/,/
1 CR*IOL
2 CR-4L 3
CR+lR
6.8X10' +-I,-
-,,;b
8.0X104 Z.ZXI05
4 CR-3R
9.0x10’
5 CR-11R
4.0x1
z-
AND FUJISAWA
0’
FIG. 2. A schematic presentation of deleted derivatives of the pacCR sequence derived from pUCAfAI-CR. Numbering is the same as described in the legend to Fig. 1. A box and an underline represent the terminally redundant and its flanking sequences in the pacCR, respectively. The horizontal arrows indicate remaining DNA in deletion derivatives of thepacCR sequence. The transducing activity was assayed and is represented as described in the legend to Fig. 1.
Phages and transducing particles were subjected to CsCl equilibrium density gradient centrifugation and the fractions from the gradient were analyzed for amp’ transformants. As shown in Fig. 3A, amp’ transductants from pUAfAI-CR yielded a single peak, banding at a slightly higher density than the phage particles, although in a broader distribution. On the other hand, amp’ transformants from pUCAfAI-CL yielded two peaks, banding at slightly higher (fractions 6, 7) and lower (fractions 10, 11) densities than phage particles (Fig. 3B). To determine the ends of packaged DNA, DNA was prepared from transducing particles purified through CsCl equilibrium density gradient. The left or right end was determined by primer extension using an appropriate 32P-labeled primer as described under Materials and Methods. With pUCAfAI-CR (Fig. 4A), the sequence toward the right ended at 5’-TGTGTCCCT-3’, corresponding to the right-end sequence of mature T3 DNA (see Fig. 2) but no unique band corresponding to the left end was detected (data not shown). With pUCAfAI-CL (Fig. 4B), the sequence toward the left ended at 5’-TCTCATAGTT-3’, corresponding to the left end of mature T3 DNA (Fujisawa and Sugimoto 1983) but no unique band corresponding to the right end was detected (data not shown). Judging from the density distribution, the transducing pUCAfAI-CR and pUCAfAICL particles appear to contain plasmid oligomers with sizes of headful length (about 40 kb; Hashimoto and Fujisawa, 1988). These results indicate that DNA in pUCAfAI-CR or pUCAfAI-CL transducing particles is plasmid oligomer and has the same right or left end as that of mature T3 DNA and a heterologous, nonunique left or right end, respectively.
of transduction
on promoter
activity
As shown above, a region containing a T3 RNA polymerase promoter, located at 98.0% genomic position, was essential for efficient transduction of recombinant plasmids. To analyze the relationship between the promoter and the DNA packaging activities more directly, a series of deletion mutations were constructed that extend into the 56-bp Aflll-Alul region in pUCAfAI-TR (containing a promoter for T3 RNA polymerase) from either the right or the left, as follows. An AfAl fragment from pUCAfAl CR, in which the fragment was flanked by HindIll and Pstl sequences, was cloned between HindIll and Pstl sites of pUC18 (pUCAfAI). The AfAl sequence was deleted rightward or leftward by mild exonuclease III digestion from the /-findIll site after Hindill digestion or from the BamHl site after BamHIKpnl double digestion of pUC AfAl and circularized after adding HindIll or Kpnl linkers, respectively. Deletions were confirmed by DNA sequencing. The TR fragment was inserted into appropriate deleted plasmids and recombinant plasmids carrying the TR se-
I
1
I
I
I
I
5
10
15
5
10
15
Fraction
J
No.
FIG. 3. Density gradient centrifugation of pUCAfAI-CR and pUCAfAI-CL transducing particles. NG30 cells carrying pUCAfAI-CR (A) or pUCAfAI-CL (B) were infected with T3 33. After incubation for 60 min, cells were collected and lysed with chloroform. Transducing particles were concentrated by two cycles of low- and high-speed centrifugation and subjected to CsCl equilibrium centrifugation and plaque-forming units on Rl 1S (su+) (0) or amp’transducers (0) were measured with all fractions as described under Materials and Methods. Fractions are left to right corresponding to from bottom to top.
DNA SEQUENCES
NECESSARY
FOR PACKAGING
ACGTN
ACGTN
BACTERIOPHAGE
793
T3 DNA
sensus sequences for T3 RNA polymerase (R + 7, +3, and +l) while the promoter activity was not impaired until deletion of G (+l) at the start site of transcription (R + 3, +l, and -6). These results indicate that the promoter activity in the pacB sequence is necessary but not sufficient for DNA packaging.
B
DISCUSSION
/
In a previous paper (Hashimoto and Fujisawa, 1988) we showed that a recombinant DNA carrying El and TR sequences from T3 concatemer DNA can efficiently be packaged into transducing particles. We used this in viva plasmid transduction as an assay system to define the minimal sequences necessary for DNA packaging (pat sequence). The TR sequence contains the target sequences for the initiation and termination cleavages, pacCR and CL, respectively. Unexpectedly, deletion experiments demonstrated that pacCR was essential but pacCL was unnecessary for efficient packaging of ,plasmid DNA. pUCAfAI-CR transducing
7 FIG. 4. The end of DNAfrom pUCAfAI-CR or pUCAfAI-CL transducing particles. Sanger’s chain termination method was used. from Ml 3 primer M4 or MV directly on circular pUCAfAI-CR (A) or pUCAfAI-CL (B), respectively. The sequence of the template is shown. The region near expected length of the mature end is shown because no other prominent bands were seen. Lane N shows the position of the end of the template DNA. (A) The sequence toward the right-end of pUCAfAI-CR was determined using 32P-labeled Ml3 primer (M4) which anneals to pUCl8 sequence left of the HindIll site. Numbering is the same as described in the legend to Fig. 1. (B) The sequence toward the left-end of pUCAfAI-CL was determined using 32P-labeled Ml3 primer (MV) which anneals to pUC18 sequence right of the EcoRl site. The nucleotides are numbered leftward (-) or rightward from the genetic left end.
quence in the same direction as in pUCAfAI TR were selected for further analysis. The promoter and packaging activities were determined by assaying for in vitro transcription and in vivo transduction, respectively, as described under Materials and Methods (Fig. 5). For deletions from the left, the efficiency of transduction began to drop as deletions entered into the consensus sequence for T3 RNA polymerase (-17 to +6) in parallel with a decrease in the promoter activity (L-l 1, L-9). For deletions from the right, the efficiency of transduction began to drop as deletions entered into the con-
L-28 L-23
4 4
L-11 L-9
4
R*l R-6
* *
FIG. 5. Relationship of the transducing efficiency to the promoter activity. The AfAl sequence of pUCAfAI-TR was deleted by exonuclease Ill from the left (L series) or the right (R series). The nucleotides are numbered leftward (-) or rightward (+) from the first nucleotide transcribed by T3 RNA polymerase. The consensus promoter sequence for T3 RNA polymerase extends from -17 to +6. An underline indicates a specific binding promoter sequence of T3 RNA polymerase. Horizontal arrows indicate deletion derivatives of the AfAl fragment. The promoter activity (0) and the transducing activity (0) were assayed as described under Materials and Methods.
794
HASHIMOTO
particles contained plasmid oligomers with the same right end as T3 and a nonunique left end. These results indicate that T3 DNA packaging machinery requires the specific sequence, pacCR, to create the DNA end essential for initiation of packaging and that the second termination cleavage can operate by a headful mechanism at the specific sequence in pacCL or at any sequence if pacCL is deleted. In the absence of pacCR, the packaging machinery would produce an end for packaging by a nonspecific cleavage at a low efficiency and cleave the pacCL sequence at the site used to form the left end of mature T3 DNA after packaging a headful of DNA (T3 DNA, 38.74 kb) (Bailey eta/., 1980), corresponding to sizes of 12 or 13 plasmid monomers (3 kb), resulting in the production of two peaks (Fig. 2B), as observed for pUCEl-TR (Hashimoto and Fujisawa, 1988). For T7 phage, Chung and Hinkle (1990b) report plasmids which contain either the pacCL or CR are packaged at about 10% of the efficiency of those with both sequences. The residual packaging of T7 plasmids results from regeneration of pacCL or CR by recombination with replicating T7 DNA or recognizing a site in pBR322 (Chung and Hinkle, 1990a,b). For T3 phage, the indispensability of pacCR appears to be unmasked because T3 3-, which was used as a helper phage in the experiment, was defective in recombination. Our experiments demonstrate that pacCR can be defined to be a 21 -bp region (R + 10 to R - 1 1) flanking the cleavage site, generating the right end. A short sequence CCTAAAG or its variants, which are repeated many times on each site of and within the terminally redundant sequence in both T3 and T7 (Dunn and Studier, 1983; Yamada et al. 1986), can be deleted without affecting the efficiency of plasmid packaging by both T3 (this paper) and T7 (Chung and Hinkle, 1990b). In a previous paper (Fujisawa et al., 1990) we showed that the cleavage at the left end of the terminally redundant sequence in pacCL did not require ,oacCR in the defined in vitro system. In in viva packaging/transduction system, the cleavage at the right end of the terminally redundant sequence in pacCR occurred efficiently in the absence ofpacCL (Fig. 1, lanes 5 and 10). These results argue against a model for DNA processing of T3IT7 phage in which DNA maturation requires both ends of the terminally redundant sequence in pacC (Watson, 1972). We have defined the functional element in the El sequence to be a 34-bp sequence, corresponding to the consensus promoter sequence protected by T3 RNA polymerase (-21 to +l 1) in the presence of GTP or GTP + ATP (Basu and Maitra, 1986). Deletions of the upstream sequence from the left decreased both the promoter and the transducing activities in parallel
AND FUJISAWA
(Fig. 5). On the other hand, deletions of the downstream sequences from the right impaired the transducing activity before the promoter activity (Fig. 5). These results indicate that the transcriptional activity is necessary but not sufficient for DNA packaging. The conclusion is consistent with the observation that transcription is required but the T7 promoter alone is not active for plasmid transduction by T7 (Chung and Hinkle, 1990b). A small viral (Guo et a/., 1987) or nonviral (Donate and Carrascosa, 1991) RNA is required for DNA packaging by $29 or X phage, respectively. However, DNA packaging of T3 phage is not inhibited but rather stimulated by RNase A treatment both in the defined or crude in vitro DNA packaging system. It seems likely that, instead of a specific RNA transcript, it is the act of transcription itself that is required for concatemer processing and packaging of T3 DNA. Although T3 RNA polymerase initiates transcription depending on a sequence -21 to +1 G (Fig. 5, R + l), the packaging requires at least transcription of a following GA stretch (+2A to +I 2G). A strong T3 promoter, 410, lacking a GA stretch, did not work as pacB (data not shown). The binding of T3 RNA polymerase becomes more stable when the GA stretch is transcribed in the presence of both ATP and GTP (Basu and Maitra, 1986). The packaging machinery may land on the pat signal with a directionality through interaction with a stable T3 RNA polymerase-promoter complex. A direct interaction between RNA polymerase and a packaging protein, gpl9, is suggested for T7 (Studier, personal communication). Although DNA negative mutations of T3 are not deficient in morphogenetic proteins (Fujisawa eta/., 1978) transduction requires gene products involved in DNA replication (Hashimoto and Fujisawa, 1988). However, plasmid DNA did not amplify replication of a short specific sequence(s). With T7, only the promoters associated with DNA replication are effective in DNA packaging (Chung and Hinkle, 1990b) and DNA synthesis is required for the production of the left-hand terminally redundant sequence during DNA packaging (White and Richardson, 1987). The relationship of transcription and DNA replication to DNA packaging remains to be elucidated. ACKNOWLEDGMENTS We are grateful to Dr. Michael Feiss (The University of Iowa) for his invaluable help with the manuscript. This work is supported by the Mitsubishi Foundation and a grant-in-aid for scientific research from the Ministry of Education of Japan.
REFERENCES BAILEY, J. N., DEMBINSKI, D. R., and MCALLISTER,
W. T. (1980). Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA. J. Viral. 35, 176-l 83.
DNA SEQUENCES
NECESSARY
FOR PACKAGING
BEHNISH,W., and SCHMIEGER,H. (1985). ln v&o packaging of plasmid DNA oligomers by Saimoneiia phage P22 independent of the pat site, and evidence for the termination cut in virro. Virology 144, 310-317. BLACK, L. W. (1981). The mechanism of bacteriophage DNA encapsidation. f’rog. C/in. Biol. Res. 64, 97-1 10. BLJKHARI,A. Z., FROSHAUER,S., and BOTCHAM, M. (1976). Ends of bacteriophage Mu DNA. Mafure (London) 264, 580-583. BASU, S:, and MAITRA, U. (1986). Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleotide triphosphate (GTP). i. Mol. Bioi. 190, 425-437. CASJENS,S., and HUANG, W. M. (1982). Initiation of sequential packaging of bacteriophage P22 DNA. J. Mol. Biol. 157, 287-298. CHEN, E. Y., and SEEEURG,P. H. (1985). Supercoil sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4, 165170. CHUNG, Y. B., and HINKLE, D. C. (1990a). Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection. J. Mol. Biol. 216, 911-926. CHUNG, Y. B., and HINKLE, D. C. (1990b). Bacteriophage T7 DNA packaging. II. Analysis of the DNA sequences required for packaging using a plasmid transducing assay. J. Mol. Biol. 216,927-938. DONATE, L. E., and CARRASCOSA,J. L. (1991). Characterization of versatile in vitro DNA-packaging system based on hybrid lambda/phi29 proheads. Virology 182, 534-544. DUNN, J. J., and STUDIER,F. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535. EARNSHAW,W. C.. and CASJENS,S. R. (1980). DNA Packaging by the double-stranded DNA bacteriophages. Cell 21, 319-331. FEISS, M., KOBAYASHI, I., and WIDNER, W. (1983). Separate sites for binding and nicking of bacteriophage X DNA by terminase. Proc. Nat/ Acad. Sci USA 80, 955-959. FUJISAWA,H., KIMURA, M., and HASHIMOTO, C. (1990). In vifro cleavage of the concatemer joint of bacteriophage T3. \/iro/ogy 174, 26-34. FUJISAWA,H., MIYAZAKI, J., and MINAGAWA, T. (1978). in v&-o packaging of phage T3 DNA. Virology 87, 394-400. FUJISAWA,H., and SUGIMOTO, K. (1983). On the terminally redundant sequences of bacteriophage T3 DNA. Virology 124, 251-258. FUJISAWA,H., and YAMAGISHI, M. (1981). Studies on factors involved in in vitro packaging of phage T3 DNA. Prog. C/in. Biot. Res. 64, 239-252. FUJISAWA,H., YONESAKI, T., and MINAGAWA, T. (1985). Sequence of the T4 recombination gene, UVSX, and its comparison with that of the recA gene of Escherichia coli. Nucl. Acids Res. 13, 74737481. GEORGE, M., and BUKHARI, A. (1981). Heterogenous host DNA attached to the left end of mature bacteriophage Mu DNA. Nature (London) 292, 175-l 76. Guo, P., ERICKSON,S., and ANDERSON, D. L. (1987). A small viral RNA is required for in v&o packaging of bacteriophage phi-29. Science 236, 690-694.
BACTERIOPHAGE
T3 DNA
795
HAMADA, K., FUJISAWA,H., and MINAGAWA, T. (1986a). Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging. Virology 151,ll O118. HAMADA, K., FUJISAWA,H., and MINAGAWA, T. (1986b). A defined in vitro system for packaging of bacteriophage T3 DNA. Virology 151,119-123. HASHIMOTO, C., and FUJISAWA,H. (1988). Packaging and transduction of non-T3 DNA by bacteriophage T3. virology 166,432-439. KUT~ER, E., GUI-~MAN, B., MOSIG, G., and RUEGER,W. (1990). Genomic map of bacteriophage. In “Genetic Maps” (S. J. O’Brien, Ed.), pp. 1.24-l .51. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MURIALDO, H.. and BECKER,A. (1978). Head morphogenesis of complex double-stranded DNA bacteriophage. Microbial. Rev. 42, 529-576. NAKASU, S., FUJISAWA,H., and MINAGAWA, T. (1983). Role of gene 8 product in morphogenesis of bacteriophage T3. L/irology 127, 124-133. RABKIN,S. D., and RICHARDSON,C. C. (1988). Initiation of DNA replication at cloned origins of bacteriophage T7. J. Mol. B/o/. 204, 903916. RAMSEY, N., and RITTCHIE, D. A. (1983). Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophageT1 DNA packaging. Nature (London) 264, 264-266. ROBERTS,S., SHELDON, R., and SADOWSKI, P. D. (1978). Genetic recombination of bacteriophage T7 in vitro. IV. Asymmetry of recombination frequencies caused by polarity of DNA packaging. tirology 89, 252-261. SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat/. Acad. Sci. USA . 74, 5463-5467. SHIBATA, M., FUJISAWA,H., and MINAGAWA,T. (1987). Early events in a defined in vitro system for packaging of bacteriophage T3 DNA. Virology 159, 250-258. STERNBERG,N., and COULBY, J. (1987). Recognition and cleavage of the bacteriophage Pl packaging site(pac). Il. Functional limits of pat and location of pat cleavage termini. 1. Mol. Biol. 194, 469479. STREISINGER,G., EMRICH, J., and STAHL, M. M. (1967). Chromosome structure in phageT4. Ill. Terminal redundancy and length determination. Proc. Natl. Acad. Sci. USA. 57, 292-295. WATSON, J. D. (1972). Origin of concatemeric T7 DNA. Nature New Biol. 239, 197-201. WHITE, J. H., and RICHARDSON, C. C. (1987). Processing of concatemers of bacteriophage T7 DNA in vitro. J. Biol. Chem. 262, 8851-8860. Wu, R., and TAYLOR, E. (1971). Nucleotide sequence analysis of DNA. Il. Complete nucleotide sequence of the cohesive ends of bacteriophage X DNA. J. Mol. Biol. 57, 491-51 1. YAMADA, M., FUJISAWA,H., KATO, H., HAMADA, K., and MINAGAWA, T. (1986). Cloning and sequencing of the genetic right end of bacteriophage T3 DNA. Virology 151, 350-361. YAMAGISHI, M., FUJISAWA,H., and MINAGAWA, T. (1985). Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA packaging specificity. Virology 144, 502-515.
