Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3

J. Mol. Biol. (1989) 207, 555-561

Nucleotide

Sequence and Complementation Studies of the Gene 10 Region of Bacteriophage T3

J. Patrick Condreayt, Sarah E. Wright and Ian J. MolineuxS Department of Microbiology University of Texas Austin, TX 78712, U.S.A. (Received 8 September 1988, and in revised form 8 December 1988) The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7. T3 genes T7 mutants. The DNA sequences of T3 and 9, 10 and 11 have been shown to complement T7 gene IOA are homologous, as are the amino acid sequences of the respective products. The translational shift to the - 1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible. The resulting gplOB products have completely different C termini.

1. Introduction Bacteriophage T7 is considered to be the prototype of a number of phages collectively referred to as the T7 group. There are three known groups of T7like phages that grow in B strains of Escherichia co&; phages within each group recombine with each other at high efficiency. T7 itself is a member of the largest group, which also includes 41, $11, W31, H, Y and Al 122 (Hausmann, 1988). The second group has three members (BA14, BA127 and BA1.56); these phages do not recombine with either T7 or with the sole member of the third group, T3 (Hausmann, 1988). Though the organization of the bacteriophage T3 genome is very similar to T7, the two phages recombine with each other only at low efficiency. DNA heteroduplex studies indicate that the T3 and T7 genomes are varyingly homologous over their complete lengths (Davis & Hyman, 1971). The late, or class III, genes are, in general, highly conserved in the two phages, as are parts of the class I and class II regions. The remainder of these last two regions and the molecular ends of the genomes were shown to have diverged, but to 1971) varying degrees (Davis & Hyman, The nucleotide sequence of T7 DNA has been

t Present address: Department of Molecular Genetics, Smith Kline and French Laboratories, P.O. Box 1539, King of Prussia, PA 19406, U.S.A. 1 Author to whom all correspondence should be

completely determined (Dunn & Studier, 1983; Moffatt et al., 1984) and can thus be compared with T3 DNA sequences as they are determined (McGraw et al., 1985; Yamada et al., 1986; Hughes et al., 1987; Schmitt et al., 1987; P. J. Beck & I. J. Molineux, unpublished results). To date, these data have confirmed the conclusions of the electron microscopic analysis. In general, genes that are essential for phage growth under all laboratory conditions (most class III and some class I and II genes) are highly conserved at both the nucleotide and amino acid level, whereas those genes that are only conditionally essential (some class I and class II genes) have diverged. More extreme cases of divergence occur when T7 genes have no T3 counterpart, and vice versa (Schmitt et al., 1987; P. J. Beck & I. J. Molineux, unpublished results). The most) abundant protein of the T3 or T7 phage particle is a product of gene 10. Two proteins, gplOA§ and gplOB, are encoded by gene 10 of both phages (Studier, 1972; Fig. 3 of Condreay & Molineux, 1989). A shift to the - 1 reading frame during translation of T7 gene 10 mRNA leads to the synthesis of the minor product, gplOB (Dunn & Studier, 1983). As a part of the characterization of T3 mutants that escape F-mediated restriction (Condreay & Molineux, 1989), the nucleotide sequence of the gene for the major capsid protein and neighboring genetic elements has been

4 Abbreviations used: gp9, gpl0, etc, protein product of gene 9, 10, etc; bp, base-pair(s).

addressed. 0022%2836/89/l 1055547 $03.00/0

555 0 1989 Academic Press Limited

556

J. P. Condreay et al.

3. Results and Discussion

determined. A comparison of these data with the equivalent part of phage T7 has shown that there are regions of high homology abruptly separated by regions that are not obviously related.

(a) Mapping

and genetic organization of the T3 Mbof D fragment

The Mb01 D fragment of T3 (see Fig. 1) had been studied by Basu et al. (1984) as part of their analysis of promoters for T3 RNA polymerase. The Hue111 map of the Mb01 D fragment shown in Figure 1 differs from that of Basu et al. (1984) primarily because electrophoretic analysis of the HaeIII fragments revealed that there were two fragments approximately 325 bp in size. This additional fragment necessitated a revision of the earlier Hue111 map; the promoter sequence that Basu et al. (1984) determined is in fact at position 53.7% from the left end of the T3 genome. Tt is designated 49 in Figure 1. The other promoter that Basu et al. (1984) detected in the 1160 bp HaeIII fragment is at position 55% on the genome ($10 in Fig. I) and is part of the sequence given below.

2. Materials and Methods (a) Bacteria, bacteriophage and plasmids Most bacteria and T3 phage strains were those described in an accompanying paper (Condreay & experiments were Molineux , 1989). Complementation performed using E. coli TBl that contained plasmids derived from pBR322 or pUC vectors. The efficiency of plating (e.o.p.) of amber mutants on the plasmid-free strain was less than 1O-5 that on an Su+ host. T7 strains 9am17, IQam13 and llam37 were from the collection of F. W. Studier. The pBR322 derivatives pAR436 and pAR1338, which contain T7 gene 10 (Dunn & Studier, 1983) and pAR441, which contains T7 gene 9 (Studier & Rosenberg, 1981) were also kindly provided by F. W. Studier. The T7 DNA in pAR441 was transferred to pT7/T3-18 (Bethesda Research Laboratories) to allow gene expression from a T3 promoter. The resulting plasmid is called pCKS43. The plasmid pDD5, a gift from W. T. McAllister, contains the T3 MboI D fragment (Fig. 1) cloned into the BamHI site of pBR322. The pT7/T3 derivatives, pSW2 and pSW4, contain T3 DNA under control of the vector T7 promoter; pSW3 and pSW5 contain T3 DNA under control of the vector T3 promoter. Plasmids pSW2 and pSW3 contain a 2600 base-pair DraI fragment of T3 DNA that includes genes 10, 11 and part of 12; pSW4 and pSW5 contain a 1300 bp DraI-HpaI fragment that includes gene lOA, but lacks the 3’ end of gene 1OB.

(b) Nucleotide sequence of the gene 10 region Figure 2 shows the nucleotide sequence of that strand of T3 (Luria) DNA that has the same polarity as T3 mRNA. The sequence starts at a FnuDII site just, upstream from the gene 20 promoter and ends at the first TaqI site in Mb01 A. Nucleotides 51 to 120 were determined by Bailey et al. (1983); their sequence differs from that of Figure 2 only in having a C, rather than a T, at position 57. This difference affects the third (wobble) position of a serine codon in gene 9 and probably reflects the use of T3 (Hausmann) by Bailey et al. (1983). Similarly, the Mb01 site, at positions 1481 to 1484, that is the K-A junction in Figure 1 is not present in T3 (Hausmann) strains. It is not known if this difference leads to a change in protein sequence. By homology with T7, the first 66 nucleotides in Figure 2 correspond to the C-terminal end of gp9.

(b) Cloning and nucleotide sequencing Cloning procedures were standard and generally followed those described by Maniatis et al. (1982). DNA sequencing was performed by the method of Sanger et a2. (1977) as adapted for Ml3 vectors (Schreier & Cortese, 1979). Both strands of the T3 DNA were sequenced and the sequences at adjoining fragments were confirmed

using overlapping clones. 0

50

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Figure 1. A physical map of bacteriophage the DNA that codes for gp8, gp9 and gpl0. experiments using various amber mutants fragments of the MboI D DNA are given (in

I

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A

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100

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T3 (Luria) DNA. The bars below the expanded part of the map indicate The lines at the bottom of the Figure show the results of marker rescue and cloned fragments of T3 DNA. The estimated sizes of the Hue111 bp).

Gene

CGCTATCGTA GCGATTCTGC CTACCGCCAA ATGGTAGAAC AGAAGGTTAT CGACTCTAGTTTCTAATTAACCCT~AAAGGGAGAGAC CATAGATGCC TACAATGGTTGAATCACCTGAGCACAGAAC TTTGTGGTCA CTCCCATAGG TGAAACATTG AGAACCAACT CGATTCAAGT AGTAACCAAA CTTTTCTTTA AATPAACATAAGGAGATTCAACATGGCTAACATTCAAGGC GGACAGCAAA TTGGTACTAA XXGGTAAG GGTCAGTCCG CAGCGGACAA ATTGGCGCTG TTCCTGAAAG TGTTCGGCGG TGAAGTCCTG ACGGCTTTCG CTCGCACCTC CGTGACCATG CCTCGTCACA TGCTGCGCTC TATTGCTTCT GGTAAGTCCG CACAGTTCCC TGTGATTGGT CGCACCAAAG CTGCTTACCT GAAACCGGGT GAGAACCTCG ATGACAAACG TAAAGATATC AAACACACCG AGAAGGTAAT CCACATTGAT GGCCTGCTGA CTGCGGATGT GCTGATTTAC GACATTGAGG ACGCGATGAA CCACTATGAC GTTCGCGCTG AGTACACCGC CCAGTTGGGT GAATCTCTGG CGATGGCGGCTGACGGTGCT GTACTGGCAG AACTGGCTGG TCTTGTTAAT CTGCCGGACG GCTCTAACGA GAACATTGAG GGTCTCGGTA AGCCAACCGT ACTGACTCTG GTTAAGCCTA CCACTGGCAG CCTGACTGAC CCGGTTGAGT TGGGTAAAGC GATTATTGCT CAGTTGACTA TCGCTCGTGC ATCCCTGACC AAGAACTACG TTCCGGCTGC TGATCGCACC TTCTACACCA CTCCTGACAA CTACTCTGCG ATTCTGGCTG CTCTGATGCC GAACGCAGCA AACTATCAGG CACTGCTCGA CCCTGAGCGC GGTACTATCC GTAACGTGAT GGGCTTCGAG GTGGTTGAGG TTCCGCACCT GACCGCTGGT GGTGCAGGCG ATACCCGTGA GGATGCCCCG GCTGACCAGA AGCACGCTTT CCCGGCTACT TCCAGCACTA CCGTTAAGGT TGCTCTGGATAACGTTGTGG GCCTGTTCCA GCACCGCTCT GCGGTTGGTA CGGTCZAUCT GAAAGACTTG GCTCTGGAGC GTGCTCGTCG TGCGAACTAT CAGGCTGACC AGATTATCGC TAAATATGCG ATGGGTCACG GCGGTCTGCG TCCAGAAGCT GCTGGCGCTA TCGTGCTCCC AAAGGTGTCG GAGTAATTCC CGACCCCACT GGAGTTACGT TAAGTCAAAA GACCATGACG CTTGTTGAGG GTGCATCCCG TGCCCTCACA GGAACAGTCC AGCCTTCCGA TGCGAATCAG TCGTTAAGCT GGTCGTCTTC TGCGGAAGAT GTGGCTGTAT GGWVLGCTGGAAAGGTTGTA GCCAAAGGTG TGGGAACGGC GGACATTACA GCTACTACCT CCAATGGCCT GATCGCTTCC TGTAAAGTGA TTGTTAACGC CGCAACGTCTTAAAACACGTAGTGCCCAATTAGGGCATCA TGTGGCGTGA AGCACTCTCA AGCCGCGAAG TGGCACATAC GTCTGACTAT GGGGACTACC AAGAGCTACG ACTATCATGT GGGCAACCAC CCTGAAAGCT CGTTGTGATT GGGATAACAA TCTACTAATA TGCAAACCCC TTGGGTTCCC TCTTTGGGAG TCTGAGGGGT TTTTTGCTTT AACCCTCACT AACAGGAGGT AACATCATGC GCTCTTATGA GATGAACATT GAGACCGCAGAAGAGCTATC AGCCGTCAAC GACATTCTGG CTTCCATCGG TGAGCCACCA GTATCG

Figure 2. Nucleotide

557

10 Region of Bacteriophage T3

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

sequence of the gene 10 region of T3 (Luria) DNA. The sequence reads 5’ to 3’ from left to right

and has the same polarity as T3 RNAs.

Similarly, the last 90 nucleotides correspond to the N terminus of gpl 1. The last 21 amino acid residues of T3 gp9 are 71 oh identical with those of T7 gp9; the first 30 residues of T3 gpl 1 are 87 y. identical with T7 gpl 1. Gene 10 was initially defined both by homology with T7 and by the estimated sizes of the protein products. Confirmation of the correct reading frame was obtained by the sequence of the amber mutation in A2R6 (Condreay & Molineux, 1989). The gene 10 products are predicted to initiate at an AUG codon at position 223 (Fig. 2). This codon is preceded by a potential Shine-Dalgarno sequence that can form seven contiguous base-pairs with the 16 S rRNA. Gene 10 of T7 codes for two proteins: gplOA and gplOB. Amber mutants in gene 10 lack both proteins and the larger of the two, gplOB, arises by an occasional translational shift to the - 1 reading frame. This shift is thought to occur at a sequence near the 3’ end of gene IOA (Dunn & Studier, 1983). Similarly, T3 gene 10 codes for two proteins and gene 10 amber mutations affect the synthesis of both (see Fig. 3 of Condreay & Molineux, 1989). The molecular weights of T3 gplOA and gplOB can be estimated from their electrophoretic mobilities in denaturing acrylamide gels to be 36,006 and 44,000, respectively. This corresponds to N 80 additional residues in gplOB. The reading frame ahead of the predicted initiation codon of T3 gene 1OA is open

for only seven codons; readthrough of the UAA termination codon of gene 10A could add nine residues to gplOA. Neither possibility accounts for the size difference between gplOA and gplOB; the latter is therefore probably made by a shift in reading frame during translation of gene 10 mRNA. It is unlikely that gplOB is initiated upstream from gplOA with a shift into the zero (i.e. gplOA) reading frame. The - 1 frame contains several stop codons and the + 1 frame could provide only 29 residues in addition to those of gplOA. A shift to the + 1 reading frame near the 3’ end of gene 1OA results in 17 additional residues, whereas a shift to the - 1 frame would result in a product 86 residues longer than gplOA. The last mechanism is most probable since it agrees best with the estimated size of gplOB and, in addition, T7 gplOB is also synthesized by a shift to the - 1 frame (Dunn & Studier, 1983). If T3 gplOB is synthesized in this way, it would be initiated at an AUG codon (position 223, Fig. 2) and would be terminated at a UAA codon at position 1521. In order for T3 gplOB to be made, the shift to the - 1 frame must occur within the last 25 codons of gene 10A (positions 1192 to 1266, Fig. 2); in T7 it was shown to occur in the last nine codons (Dunn 6 Studier, 1983). Within these last nine codons there is no DNA sequence similarity between T3 and T7. However, immediately upstream from this region the two genes are very similar (87 y. identity for 60

J. P. Condreay et al.

558 (cl)

KLRLFLK UFGGEULTRF ARTSU KLRLFLK UFGGEULTRF RRTSU PGENL ODKRKDIKHT EKUl&DGLL PGENL DDKRKDIKHT EKUI&DGLL LGESL RHAADGAULAELRGLjJNLi'D LGESL AllAADGRULA E#RGLkNijtiS LTD':U$ LGKflIIRbLT JARR$LTKNV LTDF ua LGK~~IR~LT ~~RRR:ALTKNv RRNYij R&DPERGiI RNUIIGFEUUE RRNYIj RL'rDPEKGC+IRNWIGFEUUE A$&$ @KURFDNU VGLF~~HRSRU ~Ii&f! #~UKUFIKDNU(GLF~~HRSRU

13 T7

UPHLTRGGRG UPHLTRGGRG

T3 T7

,.; .., GTUKLgDLRL ERRRRRN,QR DQllRKVRIlG HGGLRPERRGR@L*pKU$E GTUKLBDLRL ERRRRRN,QR DQllRKVRtlG HGGLRPERRGRUU$KU:E 8

Figure 3. (a) The predicted amino acid sequence of T3 gplOA compared to that of T7 gplOA. Hyphens indicate gaps that have been introduced to maintain an optimal alignment. The asterisks indicate the point where a shift in reading frame is predicted to alter the sequence of each protein. (b) The C-terminal extensions of T3 and T7 gplOB proteins, beginning at the amino acid predicted to be inserted at the site of frameshifting. bases). If frameshifting occurs by the same process in both T7 and T3 gene 10, this region of conserved nucleotide sequence may be significant. It seems likely that the frameshifts occur at equivalent positions in T7 and T3 gene 10. Dunn & Studier (1983) proposed that the shift occurs at the T7 sequence U-UUC-AAA, when a phenylalanine tRNA slips at the UUC codon and reads UUU. At the corresponding position of T3 gene 10 is the sequence C-CCA-AAG. During translation of T3 gene 10 a proline tRNA could slip and read CCC or a lysine tRNA could slip and read AAA. Either possibility could lead to gplOB synthesis. However, studies on ribosomal frameshifts during translation of the RNA phages suggest an alternative mechanism (Dayhuff et al., 1986). tRNA Thr3, which normally responds to ACCjU, can sometimes recognize the two-base codon CC. Recognition of the normal CCA proline codon by tRNA Thr3 would lead to insertion of threonine in a nascent gpl0 polypeptide. Consequently, the ribosome would only translocate two bases down the mRNA, thereby shifting into the - 1 reading frame. T3 gplOA is predicted to contain 347 residues (J& = 36,886), two more than T7 gplOA (M, = 36,550), though the two proteins are easily separated on denaturing polyacrylamide gels. T3 gplOA also contains one less basic amino acid than T7 gplOA, which accounts, at least in part, for the different electrophoretic mobilities of T3 and T7 procapsids (Serwer et al., 1983). The primary amino

acid sequence of T3 gplOA, predicted from the DNA sequence (positions 223 to 1266 in Fig. 2), is shown in Figure 3 where it is compared with T7 gplOA. The two proteins are 79% identical over their complete length, whereas the corresponding nucleotide sequences are 75.5% identical. There are six positions in Figure 3(a) where gaps have been introduced into one or the other amino acid an optimal sequence in order to maintain alignment. Assuming that each gap corresponds to a single mutation, aligning the nucleotide sequences of T3 and T7 gene IOA gives 180 changes, of which 107 are silent. More than half of the remaining 73 changes result in non-conservative amino acid computer-generated nevertheless, replacements; predictions of secondary structure indicate that T3 and T7 gplOA

are extremely

similar.

In contrast to the homology between T3 and T7 gplOA, there is no similarity in the sequence or amino acid composition of the C-terminal region of the T3 and T7 gplOB products (Fig. 3(b)). T3 gplOB is predicted to contain 443 residues (M,=45,361), 86 more than T3 gplOA, whereas T7 gplOB is only 53 residues longer than T7 gplOA. It is surprising that two genes, whose major products have been highly conserved, also code for minor, translationally frameshifted, products whose Cterminal extensions are totally different. It is equally surprising that though the specific nucleotide sequences are different, the positions in the two genes where frameshifting events most likely occur

559

Gene 10 Region of Bacteriophage T3

2" u

u C-G C-G C-G U-A U G

i

E-c" G U U G U-R ;:; C-G C=G A-U A-U

5'

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T3

5' UUG

. - 4URGCA"

RR R u t’ C-G C-G G U G=C G U G U U G u-n C-G C-G C-G C-G R-U R-U U UUUG

T7

Figure 4. Transcription termination sites for T3 and T7 RNA polymerases. Termination is at the terminal residue and potential secondary structures of the RNAs are presented.

have been conserved. This would suggest that the frameshifted products are important in phage growth. Conversely, a lack of homology between related T3 and T7 proteins is usually an indication that the proteins are not essential. In fact, the entire C-terminal extension of the gplOB proteins are not required for phage growth. Clones of gene 10 that do not contain all the information for gplOB synthesis efficiently complement gene 10 amber mutants (Dunn BEStudier, 1983; see Table 2 below). Mature particles of T3 and T7 normally contain both of their respective gene. 10 products; the 10B proteins must therefore participate in capsid assembly. However, it remains possible that a frameshifted protein of some type is required for assembly. Perhaps the role of gplOB is to provide an asymmetry to the procapsid, though the exact sequence causing this asymmetry may not be important for function. Alternatively, gplOB may have been conserved because, though not essential for eapsid formation, it enhances the assembly process or perhaps its presence extends the natural host range of the phage. The regions both 5’ and 3’ to the coding sequence of T3 gene 10 are much longer than their T7 counterparts. There are 142 bases between the predicted 5’ end of T3 gene 10 mRNA at position 82 and the initiation codon of gene 10. There is no evidence that this region is translated; a 17-residue peptide, beginning at position 96 potentially could be synthesized and, if made, would not have a T7 counterpart. The remainder of the region seems to be devoid of sites for the initiation of translation. There are also no open reading frames that are likely to be translated in the 204 bases that lie between the predicted termir@ion codon of gplOB and the 3’ end of the mRNA at position 1726. About 40 bases of this region comprise the

transcriptional terminator (see below), but remaining 162 bases have no obvious function.

the

(c) Termination signal for T3 RNA polymerase and a new promoter It has been reported that a termination signal for T3 RNA polymerase is located just after gene 10. RNA, made in vitro, terminates at a G (position 1726) that follows six uridine residues (Majumder et al., 1979). A stem-loop structure can be drawn from the RNA sequence immediately upstream from this G; this structure is very similar to the terminator for T7 RNA polymerase (Dunn t Studier, 1983). Both potential terminator structures are shown in Figure 4. T3 and T7 RNA polymerases recognize both the homologous and the heterologous terminator in vitro (Beier et al., 1977). Since the sequences of the two loops are different, information for transcription termination likely resides in the long base-paired stems of these potential structures. No T7 promoter lies between the transcriptional terminator T$J and the 413 promoter. Transcription of T7 genes 11 and 12 must therefore result from inefficient termination at T4 (Dunn & Studier, 1983). In contrast, a T3 promoter, 611, lies between T4 and T3 gene II (position 1728 to 1749). The presence of a promoter at this position suggests that, in vivo, termination of transcription at T#J might be more efficient in T3 than in T7. The sequence of the 411 promoter is shown in Table 1 together with the other known T3 promoters (Schmitt et al., 1987, and references cited therein). $111 differs from the consensus sequence at several positions and has the greatest homology with the class II promoters 41.3 and 41.5. All three are predicted to initiate transcription at an A rather Table 1 Promoters for T3 RNA polymerase Promoter

T3 units

Nucleotide

sequence

1.2

-10 +1 ATT+ACCCTCACTAAAGGGAi+

qu.05

14.3

ATTAACCCTCACTAA&SGGAGA

Cp1.1

14.7

~TTAACCCTCACTAA~GGGAGA

41.3

16.1

AHTAACCCTCACTAA6AGGAGA

WL

$1.5

19.5

ATTAACCCTCACTAA?AGGAGA

42.5

22.8

AAT’!ACCCTCACTAAAGGGAi(!

46.5

44.5

ATTAACCCTCACTAAAGGGAAb

49

51.5

AAT+ACCCTCACTAAAGGGAGA

410

54.5

ATTAACCCTCACTAAAGGGAGA

$11

57.5

+TTAACCCTCACTAA%GGAG~

413

64.8

ATTAACCCTCACTAAAGGGAGA

WR

98.0

ATTAACCCTCACTAAAGGGAGA

T3 consensus

ATTAACCCTCACTAAAGGGAGA

Asterisks denote changes from the consensus sequence.

560

J. P. Condreay et al. Table 2 Complementation of T3 and T7 mutants

Plasmid

Genes

Promoterst

pCKS43

WV

$9+#10

pDD5

WV

49 + 410 (T3); unknown§

psw2

IOA + 8, 11 (T3)

411 (T3); vector (T7)

psw3

1OA +B, 11 (T3)

411 (T3); vector (T3)

pAR436 pAR1338 psw4 psw5

10A + B (T7) 10A (T7) 1OA (T3) 10A (T3)

410 (T7); T7 DNA11 $40 (T7); T7 DNA11 Vector (T7) Vector (T3)

(T7); vector (T3)

Tester phage

e.o.p.$

T3 T7 T3 T7 T7 T7 T3 T3 T3 T3 T7 T3

6.46 6.43 6.84 0.70 1.23 0.85 0.97 675 0.96 0.97 0.94 0.92

9umHM56a 9am17 9amHM56a 9am17 lOam llam37 lOamHMlOla llamNG14a IOamHMlOla lOamHMlOla loam13 IOamHMlOla

t Only promoters that lead to gene expression are listed. Promoters with the prefix 4 are natural phage promoters; synthetic phage promoters on the vector are listed. $ Ratio of the titer on an Su- host containing the plasmid to the titer on an Su+ host. e.o.p., efficiency of plating. 5 Transcription from an unknown E. edi RNA polymerase promoter also causes phage gene expression (see the text.). 1)A minor E. coli promoter also causes gene 10 expression (Rosenberg et al., 1987).

than the more usual G residue. Class II promoters are generally thought to be weaker than class III promoters and an initial analysis has shown that 411 has only weak promoter activity in vitro (U. Maitra, personal communication). The placement of a class II-like promoter in the class III region of the genome might ensure relatively low levels of transcription of genes 11 and 12, whose products are not normally synthesized at particularly high levels in the infected cell. (d) Heterologous complementation studies Plasmid pAR441 contains T7 gene 9 under control of both T7 $9 and 410. This plasmid can complement T7 gene 9 mutants (Studier & Rosenberg, 1981) but it does not complement T3 strains (data not shown). This is probably due to inadequate synthesis of T7 gp9 in a T3-infected cell. When the T7 DNA of pAR441 was placed under T3 promoter control in a pT7/T3 vector (pCKS43), T3 9amHM56a was efficiently complemented (Table 2). Therefore, T7 gp9 can serve as the scaffolding protein for the assembly of a T3 capsid. Plasmid pDD5 contains all of T3 gene 9, cloned into the BamHI site of pBR322. Both T3 9amHM56a and T7 9am17 are complemented in strains that carry pDD5 (Table 2); the high plating efficiency of the T7 mutant is due to complementation because more than 99% of the T7 progeny were shown to retain the amber mutation. Like T7 gp9, the T3 scaffolding protein can complement a defect in the heterologous phage. T3 RNA polymerase can initiate transcription at the natural phage promoters (T3 $9 and $10) on pDD5 to give gene 9 RNAs. These promoters are unlikely to be utilized efficiently by T7 RNA polymerase, and it is likely that a vector promoter or a fortuitous E. coli promoter in the T3 DNA upstream from gene 9 allows gp9 synthesis prior to phage infection.

only the

complementation can also be Heterologous obtained using clones of gene 10 and gene 11. Plasmid pSW2 contains T3 genes 10 and 11 under T7 promoter control and complements both T7 IOam13 and T7 11am37 (Table 2). Similarly, a T3 gene 10 mutant is efficiently complemented by pAR436 and pAR1338 (Table 2). These two plasmids do not contain a T3 promoter; however, a weak promoter for E. coli RNA polymerase has been identified near the T7 410 promoter in pAR436 and pAR1338 that expresses T7 gene 10 (Rosenberg et al., 1987). Plasmid pAR1338 does not contain the 3’ end of T7 gene 10; T7 gplOA can be synthesized and a frameshifted product is expected to be made, though it does not include much of the normal C-terminal extension of gplOB. Similarly, pSW4 and pSW5 do not contain the entire sequence of T3 gene 10 and do not synthesize the complete C-terminal extension of T3 gplOB. Nevertheless, each plasmid is capable of complementing gene 10 mutants (Table 2). T7 gplOB is not required for growth of T7 (Dunn & Studier, 1983) and T3 gplOB also appears to be dispensable for T3 growth. Furthermore, the complete C-terminal extensions of the frameshifted proteins are not even essential for encapsidating heterologous phage DNAs, since clones that cannot make an intact gplOB still complement (Table 2). All ( >99%) of the progeny phage from heterologous complementation experiments were shown to retain the amber mutation. Thus in each case, complementation, and not recombination, was responsible for allowing phage growth. Therefore, T3 genes 9, 10 and 11 can substitute for their T7 counterparts in the growth of T7. Similarly, T7 genes 9 and 10 function in place of their T3 homolog during growth of T3. Consequently, proheads that contain both T3 and T7 proteins can be assembled in vivo and can encapsidate DNA of either phage. Phage tails can also be made using a mixture of T3 and T7 proteins and these can attach

Gene 10 Region of Bacteriophage T3 to a mature head. It is remarkable that, in phage assembly pathways involving complex interactions between several proteins, or between proteins and DNA, it is possible to substitute a single component with one derived from a phage that is genetically distinct. The heterologous complementation by T3 and T7 gene 10 is particularly noteworthy. The primary sequences of T3 and T7 gplOA differ by 21% but the two proteins are interchangeable. Therefore, interactions of the capsid protein with any other component of a phage assembly pathway are not critically affected even when one of every five amino acids is altered. Though there are a number of differences in the physical properties of T3 and T7 particles (Serwer et al., 1983), chimeras containing both T3 and T7 proteins are easily made. These phenotypically mixed phages have normal titers in lysates and they appear to adsorb and to eject their DNA normally. We are grateful to Drs F. W. Studier and W. T. McAllister for providing the T7 mutants and plasmids used in part of this work. We also thank Dr F. W. Studier for helpful comments. This study was supported by Public Health Service grant GM32095 from the National

Institutes

of Health.

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