Isolation of Transcriptionally Active Mutants of T7 RNA Polymerase that do not Support Phage Growth

JMB—MS 615 Cust. Ref. No. GO 014/95

[SGML] J. Mol. Biol. (1995) 250, 156–168

Isolation of Transcriptionally Active Mutants of T7 RNA Polymerase that do not Support Phage Growth Xing Zhang and F. William Studier* Biology Department Brookhaven National Laboratory, Upton NY 11973, USA

*Corresponding author

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8 –9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time. Keywords: T7 RNA polymerase; T7 lysozyme; mutants; replication; packaging

Introduction Bacteriophage T7 RNA polymerase is a single subunit enzyme of 99 kDa that catalyzes transcription from highly specific promoter sequences (Chamberlin et al., 1970; Dunn & Studier, 1983; Moffatt et al., 1984). It is the best studied representative of a family of RNA polymerases whose members extend at least from bacteriophages to mitochondria and mitochondrial plasmids (Masters et al., 1987; Oeser & Tudzynski, 1989; McAllister & Raskin, 1993). The three-dimensional structure of T7 RNA polymerase is homologous to the polymerase domain of the Klenow fragment of Escherichia coli DNA polymerase I, HIV reverse transcriptase, and perhaps rat DNA polymerase b (Sousa et al., 1993; Pelletier, Abbreviations used: IPTG, isopropyl-b-D-thiogalactopyranoside; aa, amino acid(s). 0022–2836/95/270156–13 $08.00/0

1994; Steitz et al., 1994), reflecting conserved elements of primary sequence found among all single-subunit DNA and RNA polymerases (Delarue et al., 1990). T7 RNA polymerase has been cloned, overexpressed and purified in large quantities (Davanloo et al., 1984; Tabor & Richardson, 1985), and extensive biochemical studies have yielded a rich body of information about the transcription process, including promoter recognition, chain initiation, elongation and termination (Ikeda & Richardson, 1986, 1987; Gunderson et al., 1987; Martin & Coleman, 1987; Martin et al., 1988; Sastry & Hearst, 1991; Sousa et al., 1992; Macdonald et al., 1993, 1994). Mutational analysis is beginning to define regions of sequence or structure that are involved in the various aspects of transcription (Mookhtiar et al., 1991; Gross et al., 1992; Patra et al., 1992; Ikeda et al., 1993; Rechinsky et al., 1993; Raskin et al., 1993; Bonner et al., 1994a,b; Osumi-Davis et al., 1994).

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However, T7 RNA polymerase is not simply a transcription enzyme. It participates in other processes essential for T7 growth, including entry of T7 DNA into the cell (Moffatt & Studier, 1988), initiation of replication (Fischer & Hinkle, 1980; Romano et al., 1981; Dunn & Studier, 1983) and packaging of DNA into phage particles (Chung & Hinkle, 1990; J. J. Dunn & F.W.S., unpublished results). An interaction with T7 lysozyme provides a feedback mechanism to shut off late transcription but also appears to have a role in replication and perhaps in controlling lysis (Moffatt & Studier, 1987; Cheng et al., 1994). We wished to illuminate these other roles and interactions of T7 RNA polymerase. Our approach was to randomly mutate the cloned gene for T7 RNA polymerase (T7 gene 1) and select mutants that are unable to support the growth of a T7 strain that lacks its own RNA polymerase gene. We expected to isolate mostly mutants that are deficient in transcription but, by screening for mutants that retained transcription activity, to identify new types of mutants deficient in other activities. To be able to locate mutations readily in the 2.7 kbp coding sequence, we developed a genetic mapping procedure that can localize any lethal mutation to a region small enough to be covered by a single DNA sequencing reaction.

produces new phage particles that rapidly kill all of the nearby cells in the incipient colony. Because of the limitation on the number of cells per plate, the mutation frequency must be relatively high if mutants are to be easily isolated. We chemically mutagenized pAR1219 plasmids and transferred them to host cells that had not been exposed to mutagen. Almost all of the colonies that grew in the presence of 4107 contained polymerase mutants. A few strains were excluded as presumptive host range mutants because they did not support plaque formation by wild-type T7. Comparison of the number of colonies obtained in the presence or absence of 4107 indicated that approximately 1% of the plasmids isolated from a culture treated with N-methyl-N '-nitro-N-nitrosoguanidine at a concentration of 5 mg/ml, and 2% of plasmids treated at 10 mg/ml, were defective in the polymerase. A total of 252 mutants were isolated, numbers X1 to X235 from the culture treated at 10 mg/ml and X236 to X252 from the culture treated at 5 mg/ml. Mutants X133 to X252 were isolated by direct plating of the transformation mixture and should represent independent mutations; X1 to X132 were isolated after overnight growth of the transformation mixture, but few if any appeared to be duplicates.

Results

Genetic mapping of RNA polymerase mutations in the cloned gene

Isolation of T7 RNA polymerase mutants To select T7 RNA polymerase mutants that have lost the ability to support the growth of T7 phage, cells that supply T7 RNA polymerase from the cloned gene were plated in the presence of the T7 deletion mutant 4107, which lacks the gene for T7 RNA polymerase, is unable to acquire it by homologous recombination with the cloned gene, and whose growth is absolutely dependent on T7 RNA polymerase supplied by the host cell. A small fraction of cells carrying a mutant polymerase can be selected from a vast majority of cells supplying functional polymerase by plating about 106 or fewer total cells in 2.5 ml of top agar in the presence of about 107 infectious particles of 4107. The cell concentration cannot be much higher or 4107 growth on the cells that supply functional RNA polymerase will produce enough phage to kill all cells on the plate except adsorption mutants. The concentration of 4107 must be low enough that most cells on the plate divide at least once before encountering a phage particle but high enough that every nascent colony will eventually be infected. Under these conditions, cells that cannot support the growth of 4107 form colonies because, although any infected cell is killed, no progeny phage are produced to kill nearby cells, which outgrow the capacity of phage particles diffusing from elsewhere to kill them. On the other hand, cells that do support the growth of 4107 are not able to form a colony because any cell infected

The large size of T7 RNA polymerase gene (2.7 kb) makes it difficult to locate unknown mutations in the cloned gene. Therefore, we developed a procedure for rapid genetic mapping of these lethal mutations, first to large segments and ultimately to intervals small enough to be covered by a single DNA sequencing reaction. This procedure not only locates mutations rapidly, but confirms that the mutation identified in the nucleotide sequence is responsible for the lethality. Conventional deletion mapping could not be applied to the cloned T7 RNA polymerase mutants for two reasons. Most of the phage deletions we had available ended outside the region of homology with the cloned gene and therefore could not be repaired by genetic recombination with the clone. Furthermore, functional T7 RNA polymerase is needed for recombination with the plasmid (Studier & Rosenberg, 1981), presumably to transcribe other T7 genes that are essential for recombination. To overcome these problems, we used two overlapping phage deletions with endpoints within the gene, one that enters the gene from the left and the other from the right, as illustrated in Figure 1. Each pair of deletions divides the gene into three segments, a middle segment where the two deletions overlap and the end segments, which are deleted in one of the phage mutants but not the other. A recombinant phage having a complete T7 RNA polymerase gene can be generated through a three-factor cross involving both of the deletion

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Table 1. Deletions affecting T7 gene 1, in order of right end

Figure 1. Mapping lethal T7 RNA polymerase mutations by genetic recombination. A clone of T7 RNA polymerase having a lethal mutation at amino acid residue 400 is represented aligned with the DNA of T7 deletion phages 4137, 4127, 4122 and 3490. The coding region for T7 RNA polymerase is between the two vertical lines, and the cloned fragment (heavy line) extends only slightly beyond the end of the coding sequence. The portion deleted from each phage DNA is represented by a gap within parentheses, with the number of the last (or first) deleted codon given. A three-factor cross between the cloned fragment and deletions 4137 and 3490 to generate wild-type T7 is represented. Recombination between the cloned fragment and deletions 4127 and 3490 or between it and 4122 and 3490 could not produce viable phage particles because any phage that acquired gene 1 would necessarily acquire the lethal mutation. The results of the three different three-factor crosses would place the lethal mutation between amino acid residues 221 and 502.

mutants and the cloned gene in the plasmid. Viable recombinants can be formed only if the lethal mutation in the clone lies in either end segment and not if it lies in the overlapping segment. Performing the recombinations in BL21(DE2) or HMS174(DE2) supplied enough wild-type T7 RNA polymerase from the chromosomal gene to allow growth and recombination. Although viable phage can be produced by recombination with the chromosomal gene, the frequency produced by recombination with the multicopy plasmid (when viable phage can result) was usually 10 to 20 times higher, so that the mapping result was usually unambiguous. To localize a lethal mutation in a clone, deletion 3490, whose left end is in the coding sequence, was crossed with different members of a set of deletions whose right ends are at various positions in the coding sequence (Table 1). Initial mapping used three different pairs of overlapping deletions to place a mutation into one of four segments (Figure 1). Mutations of particular interest were more closely mapped by crosses involving other deletions of Table 1 and then identified by DNA sequencing. Mapping with the set of deletions in Table 1 localizes single lethal mutations, the most likely case, but does not exclude the possibility that additional lethal mutations might be present or that more than one mutation may be required for lethality. In such

Deletiona

Left

Right

Length

Repeat (bp)

aa deleted

4137 4108 4114 4120 4113 4110 4121 4127 4146 4125 4106 4122 4128 4124 4107 3490

768 932 1748 1947 1900 2529 1895 3100 692 2402 2535 2026 876 1085 1472 3342

3833 4022 4147 4163 4325 4364 4470 4674 4736 4739 4974 5253 5499 5789 5875 5881

3071 3096 2404 2222 2437 1840 2582 1581 4053 2341 2443 3235 4630 4712 4419 2538b

7 7 6 7 13 6 8 8 10 5 5 9 8 9 17 0

1-221 1-284 1-326 1-331 1-385 1-398 1-434 1-502 1-522 1-523 1-602 1-695 1-777 1-873 1-883 57-883

a Each deletion except for 3490 arose by a crossover between short repeated sequences in wild-type T7 DNA; the position given for the left end of each deletion is the last base-pair in the left copy of the repeated sequence and the right end is the first base-pair in the right copy. The length is the number of base-pairs of wild-type sequence deleted, calculated as Right − Left + Repeat −1. b 2538 base-pairs of wild-type T7 DNA were deleted and a 10 bp linker containing a BamHI site was inserted.

cases, the position of the leftmost lethal mutation or the rightmost of multiple mutations required for lethality will be determined. A set of mapping deletions having different left ends as well as different right ends within the gene could reveal these more complex cases or determine that all of the relevant mutations must lie within a single interval.

Initial classification of the mutants A few of the mutants were discarded for various reasons, and the 234 remaining mutants were placed in one of five groups (Table 2) according to their ability to produce the T7 RNA polymerase protein, their apparent transcription activity, the ability of the induced plasmid to support plaque formation by 4107, and the ability of the uninduced plasmid to support plaque formation by 4107,AK6, which carries a suppressor mutation described in a later section. Such suppression is unique to and defines group A mutants. Possible transcription activity in vivo was measured by two different tests. One was the ability of the induced plasmid to interfere with plaque formation by a l phage that carries the f10 promoter for T7 RNA polymerase: the greater the transcription activity the smaller the l plaques. The second test, applied to both uninduced and induced plasmid, was the ability to increase the chloramphenicol resistance of BL21(DCAT4), which carries the resistance gene in its chromosome under control of the f10 promoter and Tf terminator: the greater the transcription activity the greater the resistance. None of the uninduced plasmids supported plaque formation by 4107 (the basis for their selection), but many of them

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Table 2. Distribution of 234 T7 RNA polymerase mutants into groups Group A B C D E

Protein Full-length Full-length Full-length Truncated Undetected

Transcription − IPTG + IPTG + + − − −

+ + (−) + or − + or −

4107 plaques − IPTG + IPTG − − − − −

+ − (−) + or − + or −

4107,AK6 − IPTG Number + − − − −

37 2 77 63 55

Parentheses indicate that some members of the group may have some activity.

did so to a greater or lesser extent when induced: plaque formation implies at least some transcription by the mutant polymerase, but lack of plaques does not necessarily imply lack of transcription (since other essential functions of the polymerase might be affected). About half of the 234 mutants failed to produce full-length polymerase protein (Table 2), being about equally divided between those in which a truncated polymerase protein was apparent (group D) and those in which no polymerase protein was apparent (group E). These mutants were not considered further. Of the 116 mutants that produced significant amounts of full-length polymerase protein, 77 (two-thirds) appeared to be severely impaired in transcription activity (group C). A few of these mutant plasmids provided some chloramphenicol resistance, but analysis of protein synthesis after infection by 4107 showed little if any late T7 protein synthesis in one such case, X176. Perhaps some of these mutants may be similar to the linker insertion mutant ins691 described by Gross et al. (1992), which apparently can transcribe the chloramphenicol resistance gene from the supercoiled host chromosome but cannot transcribe T7 genes from the linear T7 DNA. One group C mutant, X91, provided some chloramphenicol resistance and also allowed plaque formation by 4107 in the presence of inducer. X91 is distinguished from the group A mutants by its inability to support plaque formation by 4107,AK6 and from group B mutants because it has less transcription activity yet supports plaque formation by 4107 in the presence of IPTG. Genetic mapping showed that the group C mutations are widely distributed across the gene (Table 3). They are interesting candidates for biochemical analysis of transcription. We were surprised that 39 mutants, one-third of those that produced full-length protein, showed significant, in many cases normal, transcription activity in the various in vivo tests. Two of these mutants, X92 and X134, appeared to have normal transcription activity but failed to support plaque formation by 4107 even when fully induced (group B in Table 2). They turned out to have the same mutation, and its effect is described in the next section. The remaining 37 mutants (group A in Table 2) were all able to support plaque formation by 4107 upon induction, and also supported plaque formation by the suppressor strain 4107,AK6 (Table 4).

A mutation that prevents DNA maturation and packaging The group B mutants X92 and X134 appeared to have normal transcription activity in both the l inhibition and chloramphenicol resistance tests, but even the fully induced plasmids failed to support plaque formation by 4107. Genetic mapping placed both lethal mutations in the interval between amino acids 57 and 221 of the polymerase. DNA sequencing through this region revealed that both mutants have the same C to T change at nucleotide 3687, which predicts a change of Arg173 to Cys. X134 but not X92 also has a silent C to T change at nucleotide 3854, coding for Ser228. The presence of the secondary mutation in X134 but not X92 suggests that the two mutations arose independently.

Table 3. Distribution of mutations that interfere with transcription (group C) aa 57-221 X14 X15 X16 X52 X53 X64 X81 X88 X142 X145 X148 X154 X171 X208b X225 X232

aa 221-502

aa 502-695

X54 X72 X91a,c X95a X98a X192 X209 X220a X222a X224a X234a

X11 X21 X23 X26 X33 X35 X44 X61 X65 X66 X69 X75b X93a X100 X152 X165 X176c X188 X189 X193 X197 X202 X216 X227a

aa 695-883 (or 1–57) Not mapped X27 X63 X70 X85 X96 X139 X140 X149 X150 X153 X158 X160 X161 X164 X186 X198 X210 X211 X230 X233 X237 X239

X28 X79 X181 X185

a These mutants were mapped with deletion phage 4146 rather than 4127, which gave intervals of aa 221-522 or 522-695 instead of 221-502 or 502-695. b Mapping results were ambiguous because the mutation was close to a deletion end point: X75 could be to either side of aa 695, and X208 could be to either side of aa 221. c X91 and X176 had appreciable transcription activity, and 4107 formed plaques on X91-containing cells in the presence of IPTG. Neither mutant allowed plaque formation by 4107,AK6 in the absence of IPTG.

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Figure 2. Mutants X92 and X134 are deficient in maturation of DNA from concatemers. Total DNA was analyzed every four minutes between eight and 36 minutes after 4107 infection of cultures of BL21(DCAT4) carrying pAR1219 or the X92 or X134 mutant plasmid growing at 37°C in B25P medium (which is B2 medium (Studier, 1975) containing 0.8 mM phosphate, five times the original amount), using a protocol developed by J. J. Dunn. Each 350 ml sample of infected culture was mixed with 50 ml of 80 mM Tris-HCl (pH 8.0), 80 mM Na3 EDTA, 4% (w/v) sodium dodecyl sulfate and frozen in a solid CO2 /ethanol bath, thawed, and incubated with 50 mg of proteinase K for two hours at 37°C. Then 25 ml of 4 M NaCl was added, the DNA was precipitated with 300 ml of isopropanol and redissolved in 100 ml of 10 mM Tris-HCl (pH 8.0), 0.1 mM Na3 EDTA, and 5 ml of each sample was digested with a mixture of BglI, BstNI, EcoRI, RNase A and RNase T1 . The patterns obtained by electrophoresis through an 0.8% (w/v) agarose gel followed by staining with ethidium bromide are shown, the time of sample collection increasing from left to right in each set, along with HpaI-cut T7 DNA as a standard at the far right. The positions of the left-end, right-end, concatemer junction and hairpin fragments (Chung et al., 1990) and of the largest fragment produced from the plasmid DNA are indicated. The right-end and hairpin fragments produced after 4107 infection comigrate just behind the left-end fragment in these patterns. The hairpin fragment is made in both mutants, but not the left-end or right-end fragments.

Arg173 is apparently in a surface loop (Sousa et al., 1993) that is sensitive to protease digestion (Davanloo et al., 1984; Tabor & Richardson, 1985; Grodberg & Dunn, 1988; Muller et al., 1988). Mutations in this protease-sensitive region of the polymerase have previously been found to prevent T7 DNA from being matured from concatemers and packaged into phage particles (John Dunn & F.W.S., unpublished observations) and it seemed likely that the R173C mutation would have the same effect. Indeed, both the X92 and X134 plasmids allowed normal protein synthesis and DNA replication by 4107 but neither mutant supported maturation of the phage DNA from concatemers to produce the left or right end found in phage particles (Figure 2 and not

Novel Mutants of T7 RNA Polymerase

shown), exactly the behavior of the previously characterized mutants. Clearly, the lethal effect of the R173C mutation in T7 RNA polymerase is to prevent DNA maturation and packaging. Two previously characterized polymerase mutations that prevented packaging (insertion of glycine after Lys179 or after Lys180) were weakly suppressed by mutations in a small region of gene 19 (J. J. Dunn, personal communication). A similar weak suppressor of X134 was isolated, and the suppression was determined to be due to a change of C to A at nucleotide 38,904, which predicts a change of Ala511 to Asp in the gene 19 protein. A different mutation of the same amino acid had previously been isolated as a suppressor of the glycine insertion mutants (J. J. Dunn, personal communication). These suppressors suggest that the DNA maturation and packaging process may involve an interaction between T7 RNA polymerase and the gene 19 protein, a protein known to be required for DNA maturation (Studier, 1972).

Mutants that are hypersensitive to inhibition by T7 lysozyme A feature of all group A mutant plasmids tested was that plaques arose when large numbers of 4107 were plated on an uninduced culture. The suppressor mutations that allowed 4107 growth appeared not to be allele-specific, because phages isolated on any of several different group A mutant plasmids could grow on all of them. This lack of specificity suggested that the group A mutants might have a common defect. To elucidate this defect, the X4 and X19 mutations were analyzed in detail. Genetic mapping placed them between amino acid residues 695 and 883, and 398 and 502, respectively. DNA sequencing of the plasmids in these regions found a change of G to A at nucleotide 5631 of T7 DNA, predicting a change of Ala821 to Thr in X4, and a change of G to A at nucleotide 4563, predicting a change of Ala465 to Thr in X19. Each mutation was transferred from plasmid to phage by genetic recombination during growth of wild-type T7 on a host carrying the mutant plasmid. Since induction of the X4 or X19 plasmids supports the growth of phages defective in gene 1, each mutant phage could be propagated on its corresponding induced mutant plasmid, thereby avoiding the problem of accumulating wild-type recombinants when mutant phages are propagated on the wild-type plasmid. The mutant recombinants were identified by spot testing individual plaques for inability to grow on a host without plasmid. Analysis of RNA and protein synthesis after infection of BL21 by the X4 and X19 mutant phages revealed that gene expression began normally in both infections but shut off prematurely. Incorporation of [3H]uridine was strongly depressed by the X4 mutation and even more so by X19 (Figure 3(a)). The pattern of protein synthesis showed essentially normal synthesis of early proteins and those late

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proteins specified by genes extending as far right as gene 8 or 9 on the genetic map, but synthesis of gene 10 protein and proteins specified by genes farther to the right was strongly depressed (Figure 3(b)). Shown for comparison is the pattern after infection by 4107, where the complete lack of T7 RNA polymerase (and genes 0.5, 0.6 and 0.7) means that late proteins could be expressed only by E. coli RNA polymerase transcribing past the early transcription

terminator. The major T7 proteins were the gene 0.3 and 1.3 early proteins, whose synthesis continued long past the normal time of shutoff; only small amounts of late proteins were made. All of the T7 proteins known to be needed for DNA replication were made in normal amounts at the normal times after infection by either X4 or X19 (Figure 3(b)), but accumulation of X4 DNA reached only 10% and X19 DNA only 50% of the wild-type

Table 4. Properties of group A T7 RNA polymerase mutants Mutant plasmid Wild-type No gene1 X55 X131 X82 X117 X4 X56 X191 X212 X242 X251 X60 X126 X19 X83 X76 X119 X151 X168 X170 X102 X162 X77 X109 X112 X184 X195 X215 X217 X51 X31 X59 X129 X12 X199 X49 X223 X32

Protein levela

Transcriptionb −IPTG +IPTG lf10R

++ − ++ ++ ++ ++ + ++ + +

++ − ++ ++ + ++ + ++ + +

++ − ++ + ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ + + ++ ++ + + ++ ++ + + ++ ++ ++ + ++ ++ ++ ++ ++ ++ + +

+/− + + + ++ + + +/− + + + + +/− +/− + + ++ ++ +/− + + +/− + + + + −

+ + ++ ++ ++ + + + + ++ + ++ + + + ++ ++ ++ + + + + + + + ++ +/−

0 Big 0 0 0 0 0 0 Tiny Tiny

0 0 0 0 Tiny Small Small

Small Small Small Small Small

4107 +IPTGc ++ − ++ +++ +++ +++ ++ ++ +++ +++ ++ ++ +++ + +++ +++ +++ +++ +++ + ++ +++ +++ ++ ++ +++ ++ + + + +++ ++ ++ +++ + + + + +

4107,AK6 Map location −IPTGc (aa) ++ − +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + +/− +/− +/− +/− +/− +/− +/− +/− +/−

221-502 221-502 221-522 502-695 821 695-883d 695-883d 695-883d 57-221 57-502 465 221-522 502-695 502-695 502-695 502-695 502-695 522-695 57-221 502-695 522-695 695-883d 695-883d 695-883d 695-883d 695-883d 57-221 221-502 221-502 221-502 502-695 502-695 695-883d 695-883d

The group A mutants are grouped approximately according to how well they support plaque formation by 4107,AK6; within groups they are ordered by map interval. Any blank in the Table indicates that the test was not made. a ++ indicates a level of protein comparable with that produced after induction of wild-type plasmid pAR1219; + is about quarter to half the level of wild-type. b ++, +, +/ − , − correspond to 625, 250, 125 or 50 mg of chloramphenicol added to top agar as the greatest amount on which a spot test of BL21(DCAT4) carrying the mutant plasmid would grow; BL21(DCAT4) itself grew with 50 mg but not 125 mg in top agar. The symbols also correlated with strength of growth when cultures were streaked on plates containing 25 or 12.5 mg/ml. Relative plaque sizes are given for lf10R plated on ED8739 carrying the mutant plasmid in the presence of IPTG. c Plating of 4107 in the presence IPTG and 4107,AK6 without IPTG was done in BL21(DCAT4) host containing the mutant plasmids. +++, ++, +, +/ − indicate normal, medium, small or tiny plaques. The plaques of 4107 on induced wild-type plasmid pAR1219 were smaller than normal because a high level of T7 RNA polymerase is somewhat inhibitory. Addition of the AK6 mutation reduces the plaque size of 4107 on wild-type pAR1219; mutations in the first group increase the plaque size of 4107,AK6 and those in the last two groups decrease it. d These mutations could also be located between aa 1 and 57.

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(a)

(b)

Figure 3. RNA and protein synthesis after infection by X4 and X19. A culture of BL21 grown at 30°C in M9 medium to an A600 of 0.4 was infected with wild-type, X4, X19 or 4107 phage at a multiplicity of 10. (a) Samples were labeled for one minute with 10 mCi of [5,6-3H]uridine per ml (37.6 Ci/mmol) at different times after infection; acid-precipitable radioactivity is plotted as a function of the time at which label was added to samples infected with wild type (W), X4 (t) or X19 (T). (b) Equivalent cultures were labeled for four minutes with 20 mCi [35S]methionine/ ml beginning at four minutes before infection and 0, 4, 8, 12 and 16 minutes after infection and analyzed by electrophoresis through a 10% to 20% polyacrylamide gradient gel in the presence of sodium dodecyl sulfate followed by autoradiography. The positions of several T7 late proteins are indicated, using Figure 3 of Dunn & Studier (1983) as a standard.

level (Figure 4). Since the replication proteins were all made and the only known mutation in either phage was in T7 RNA polymerase, the reduced replication suggests that T7 RNA polymerase is required for appreciable T7 DNA replication in vivo, in agreement with previous conclusions (e.g., Dunn & Studier, 1983) and in vitro results (Fischer & Hinkle, 1980; Romano et al., 1981). (An earlier result apparently demonstrating the involvement of T7 RNA polymerase in replication in vivo (Hinkle, 1980) must be discounted because the temperature-sensitive mutation thought to be in the T7 RNA polymerase gene was later found to be located elsewhere (F.W.S., unpublished results)). Concate-

Figure 4. DNA replication after infection by X4 and X19. Samples were prepared as described in the legend to Figure 2, except that the host was BL21, cultures were infected at 30°C, and samples were collected at 4, 8, 12, 14, 16, 20, 24 and 28 minutes after infection. These band patterns are different from those shown in Figure 2 because they were produced from full-length T7 DNAs rather than 4107 deletion DNA. Band intensities were measured with ImageQuantTM (Molecular Dynamics) on scanned photographic images of exposures in the linear range, as determined from the HpaI-cut T7 DNA used as standard. (The image in the Figure was overexposed to allow the X4 DNA bands to be visualized.) The maximum accumulation of X4 and X19 DNA was about 10% and 50% of wild-type T7 DNA, respectively.

mers were made by both X4 and X19 but were not processed to form the left and right-end fragments of mature T7 DNA (Figure 4), presumably because few if any proheads or DNA maturation proteins were made (Figure 3(b)). The premature cessation of RNA and late protein synthesis after infection by X4 and X19 suggested that these mutant polymerases might be hypersensitive to inhibition by T7 lysozyme, which normally binds to T7 RNA polymerase and shuts off late transcription (Moffatt & Studier, 1987). Lysozyme is the product of gene 3.5, and the time of appearance of this protein correlates well with the timing of shut-off of transcription during X4 and X19 infection. To test directly whether the X4 and X19 mutant RNA polymerases are hypersensitive to inhibition by T7 lysozyme, the mutant genes under control of the lacUV5 promoter were transferred to the chromosome of BL21, as described in Materials and Methods. Expression of b-galactosidase from a multicopy plasmid having the gene under control of the f10 promoter was measured after induction of wild-type or mutant T7 RNA polymerase in the presence of a compatible plasmid that supplies no T7 lysozyme (pACYC184), a low level (pLysS) or a high level (pLysE). The ability of the fully induced cells to form colonies was also determined as another

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Table 5. b-Galactosidase activity and colony formation after induction of mutant T7 RNA polymerases in the presence of different levels of T7 lysozyme pACYC184 Activity Colonies Wild-type X4 X19 BL21 control

100 76 71 9

pLysS Activity Colonies

− 47 − 49 − 11 (host cell, no plasmids)

− − +

pLysE Activity Colonies 25 7 7

− + +

BL21 lysogenic for a lambda derivative that placed wild-type, X4 or X19 T7 RNA polymerase in the chromosome under control of the lacUV5 promoter each carried two compatible plasmids, pAR2197, which carried b-galactosidase under control of the f10 promoter, and either pACYC184, pLysS or pLysE. Cultures were grown at 30°C in M9 medium until A600 reached about 0.6 and then made 0.4 mM in IPTG to induce the RNA polymerase. b-Galactosidase activities were measured two hours after induction (Miller, 1972) and are given relative to the value for wild-type polymerase and pACYC184, the average of three experiments. The activity induced in a culture of BL21 containing no lysogen and no plasmids is also shown. At the time of induction, samples were plated in the presence of IPTG to measure ability of the continuously induced cells to form colonies.

measure of transcription activity, as cells with a high enough level of transcription by T7 RNA polymerase are unable to grow (Studier & Moffatt, 1986; Studier, 1991). In the absence of lysozyme, induction of the X4 and X19 polymerases produced about 70% as much b-galactosidase activity as did the wild-type polymerase, and in all three cases the induced cells failed to form colonies (Table 5). The low level of lysozyme supplied by pLysS only partially inhibited b-galactosidase production by the wild-type and X4 polymerases but almost completely inhibited production by X19. This difference was also reflected in the ability of the induced X19 but not X4 or wild-type cells to form colonies. The high level of lysozyme supplied by pLysE appeared to completely inhibit both the X4 and X19 polymerases but not wild-type, as indicated both by b-galactosidase level and colony formation. These tests show that both the X4 and X19 RNA polymerases are more sensitive than wild-type RNA polymerase to inhibition by T7 lysozyme, with X19 apparently more sensitive than X4 to lower levels of lysozyme (consistent with the apparently stronger inhibition of transcription during X19 phage infection, Figure 3(a)). The suppressors of X4 and X19 are in T7 lysozyme If the X4 and X19 RNA polymerases are more sensitive to inhibition by T7 lysozyme, the suppressors of these mutations seem likely to be located in gene 3.5, which codes for lysozyme. To test this possibility, the lysozyme gene was cloned into pBR322 from each of two suppressor-carrying 4107 phages, isolated by their ability to grow on a host containing the X4 plasmid or the X19 plasmid. When 4107 itself was grown on BL21(DE2) containing gene 3.5 cloned from either of the suppressor-carrying phages, about 4% of the progeny phages carried a suppressor, an appropriate fraction if the suppressors were acquired by recombination with the

plasmid (Studier & Rosenberg, 1981). On the other hand, when 4107 was grown on BL21(DE2) containing gene 3.5 cloned from wild-type T7, only 0.03% of the progeny phages acquired a suppressor, comparable with the background level. Therefore, at least some suppressors of the group A mutations must indeed be located in the T7 lysozyme gene. The mutation responsible for suppression was identified in the nucleotide sequence of the lysozyme gene of each of 30 different suppressors isolated by plating large numbers of 4107 on a host carrying either X4 or X19. These mutations affected 16 different amino acid residues, mostly near the amino terminus of the lysozyme protein or in a region encompassing residues 30 to 42, which appear to define the surface of lysozyme that interacts with T7 RNA polymerase (Cheng et al., 1994). No suppressor was identified outside of the lysozyme gene. Several of the suppressor lysozymes were cloned and found to be less effective than wild-type lysozyme in inhibiting wild-type T7 RNA polymerase, in experiments similar to those summarized in Table 5. It seemed likely that the suppressor lysozyme proteins would bind less strongly to T7 RNA polymerase, and we confirmed this with two of them in a gel-shift assay (Figure 5). Because many of the suppressor mutations were located near the amino terminus of lysozyme, we constructed the AK6 mutant, which lacks amino acid residues 2 to 5 of the wild-type protein but retains normal amidase activity (Cheng et al., 1994). As shown in Figure 5, the AK6 lysozyme appears to be unable to bind T7 RNA polymerase. If the group A mutants are all lethal because of hypersensitivity to lysozyme inhibition, the AK6 mutation, by preventing the association of lysozyme and polymerase, should allow 4107 to grow on any of them. We constructed the 4107,AK6 double mutant to test this idea and to have a simple test for identifying this type of mutant. Indeed, the AK6 mutation permits 4107 to form plaques on uninduced plasmids of any of the 37 group A mutants, although

JMB—MS 615 164

Figure 5. Gel-shift assay of relative affinities between wild-type T7 RNA polymerase and mutant T7 lysozymes. Mixtures containing (left to right in each sample) 1.0, 0.5, 0.25, 0.125 mM or no wild-type T7 RNA polymerase and 0.2 mM of wild-type or mutant T7 lysozyme (labeled with [35S]methionine) were incubated for five minutes at room temperature in 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2 , 4 mM spermidine, 12 mM b-mercaptoethanol and 100 mg bovine serum albumin/ml before electrophoresis through a 1% agarose gel in 25 mM sodium acetate, 20 mM potassium phosphate (pH 7.7). The mutant lysozymes were R2S (Arg2 replaced by Ser), A1MD (Ala1 replaced by Met-Asp), and AK6 (deletion of amino acid residues 2 to 5, Arg-Val-Gln-Phe). In this assay (to be described in more detail elsewhere), association of the more basic lysozyme with the more acidic polymerase shifts the labeled lysozyme to the lower band in the autoradiogram. Higher concentrations of polymerase were needed to shift the R2S and A1MD mutant lysozymes, indicating lower affinities. The AK6 mutant lysozyme gave no indication of binding to T7 RNA polymerase.

only poorly on some of them (Table 4). The 4107,AK6 double mutant is unable to form plaques on mutant plasmids of groups B or C.

Discussion As expected, most of the T7 RNA polymerase mutants selected for inability to support the growth of T7 were defective in transcription, but a surprisingly high one-third of the mutants that made full-length protein (one-sixth of the total) showed substantial transcription activity in vivo. Two mutants, which turned out to carry the same mutation, had normal transcription but were defective in maturation of T7 DNA from concatemers, similar to mutants constructed previously in the same region of the gene (J. J. Dunn & F.W.S., unpublished results). Following previous work with this type of mutation (J. J. Dunn, personal communication), we isolated a suppressor in gene 19. The gene 19 protein is known to be required for DNA maturation (Studier, 1972), and finding suppressors in this gene suggests that T7 RNA polymerase may interact with the gene 19 protein in the maturation and packaging process. The remaining 37 mutants with transcription activity appeared to be hypersensitive to inhibition by T7 lysozyme, a previously unknown type of mutation that causes premature cessation of late transcription. The two mutations characterized in detail, X4 and X19, are far apart in the amino acid sequence of T7 RNA polymerase (residues 821 and

Novel Mutants of T7 RNA Polymerase

465) but the Ca atoms of the two residues are about ˚ apart in the three-dimensional structure (Sousa 16 A et al., 1993; Brookhaven Protein Databank entry 2RNP). Although side-chains are not represented in the structure, these residues seem close enough to the surface and to each other that perhaps both could interact with T7 lysozyme. Both mutations change alanine to threonine, and perhaps they increase the binding affinity and thereby account for the hypersensitivity. However, at least some hypersensitivity mutations are located in the N-terminal domain of the polymerase molecule (Table 4), which is almost certainly too far away for a bound lysozyme molecule to be able to contact all of the affected residues. The large number of mutations that confer hypersensitivity also makes it seem unlikely that all of them increase the affinity for lysozyme. Perhaps at least some of the mutations affect the transcription reaction in subtle ways that make it more susceptible to inhibition by lysozyme binding. Polymerase mutants that are hypersensitive to inhibition by T7 lysozyme made it possible to isolate large numbers of suppressor mutations in the lysozyme gene. These in turn allowed a surface on T7 lysozyme that interacts with T7 RNA polymerase to be identified (Cheng et al., 1994). Identifying amino acid changes in the polymerase that reduce its affinity to lysozyme (Moffatt & Studier, 1987) might also allow the surface that interacts with lysozyme to be identified in the structure of T7 RNA polymerase (Sousa et al., 1993). We had hoped to find transcriptionally active mutants that are defective in replication. In fact, the X4 mutant that is hypersensitive to inhibition by lysozyme made normal amounts of the T7 replication proteins at the normal time but produced only about 10% as much T7 DNA as wild-type. This may indicate that T7 RNA polymerase is directly involved in replication but may also be an indirect effect of the lysozyme inhibition. For example, entry of T7 DNA into the cell is thought to be coupled to transcription (McAllister et al., 1981; Zavriev & Shemyakin, 1982; Moffatt & Studier, 1988), and perhaps the inability to express genes to the right of genes 8 or 9 is because entry of the DNA has been halted, thereby also preventing replication. Transcriptionally active mutants that are defective in replication or have other interesting properties might be more readily isolated by modifying our selection procedure. For example, 4107 selection with the plasmids in BL21(DCAT4) in the presence of chloramphenicol should exclude mutants that are not transcriptionally active, and selection in the presence of IPTG might eliminate the large number of mutants that are hypersensitive to lysozyme inhibition (because most such mutants seem to support 4107 growth when induced). Selection with 4107,AK6 would also exclude lysozyme-hypersensitivity mutants. Perhaps such selections would yield additional interesting types of T7 RNA polymerase mutants.

JMB—MS 615 165

Novel Mutants of T7 RNA Polymerase

Materials and Methods

Isolation of T7 RNA polymerase mutants

Bacteria, phage, plasmids and growth conditions

Plasmid pAR1219 was mutated by adding N-methyl-N 'nitro-N-nitrosoguanidine (final concentration of 5 or 10 mg/ml) to a culture of BL21/pAR1219 growing at 37°C in tryptone broth plus ampicillin at an A600 of 0.5. After a further 70 minutes shaking at 37°C, cells were harvested from 10 ml of culture and plasmids were isolated as described (Studier, 1991). The isolated plasmids were used to transform frozen competent E. coli HB101 (BRL) according to the manufacturer’s instructions. Portions of each transformation mixture were plated in the presence of ampicillin to measure total transformants and in the presence of ampicillin plus the T7 RNA polymerase deletion mutant 4107 (107 infectious phage particles added to 2.5 ml of top agar) to select for mutant plasmids unable to support the growth of T7. The remaining transformation mixture was diluted 20-fold in TYG medium plus ampicillin, grown overnight, and dilutions of the resulting culture were also plated in the presence of 4107 to select for mutant plasmids. Colonies that grew in the presence of 4107 were picked into fresh medium, incubated for a few hours at 37°C, and streaked onto an ampicillin plate for isolation of a single colony. Each mutant was tested for ability to plate wild-type T7 (to exclude host range mutants that might also have been selected) and for inability to plate 4107 (to confirm the desired phenotype).

Except as noted, bacterial and phage strains, growth media, and conditions for growth and plating were as described (Studier, 1969; Studier & Moffatt, 1986). When present, ampicillin was at a concentration of 20 mg/ml in liquid culture or in plates used for streaking colonies, or 0.5 mg was added to the 2.5 ml of top agar when plating on 20 ml plates; chloramphenicol was at a concentration of 12.5 or 25 mg/ml in liquid or plates, or 125 or 625 mg was added to top agar; and isopropyl-b-D-thiogalactopyranoside (IPTG) was used at a concentration of 400 mM, or 2.5 mmol was added to top agar. The nucleotide sequence of T7 DNA and locations of its genetic elements are given by Dunn & Studier (1983) and Moffatt et al. (1984), corrected by changing GT to TG at nucleotides 11,061–11,062 (Cheng et al., 1994) and inserting T at nucleotide 17,511 (J. J. Dunn, personal communication). The corrected sequence is given in GenBank, accession number V01146. Nucleotide sequences refer to the l strand, which has the same sequence as the T7 mRNAs. Amino acid numbering begins at the initiating methionine residue for T7 RNA polymerase (Moffatt et al., 1984) and at the next amino acid residue for T7 lysozyme and the gene 19 protein (Dunn & Studier, 1983). Plasmid pAR1219 carries the gene for T7 RNA polymerase under control of the lacUV5 promoter in the BamHI site of pBR322 (Davanloo et al., 1984). DE2 and DE3 are derivatives of phage l that have the gene for T7 RNA polymerase inserted into the BamHI site of the l cloning vector D69 (Mizusawa & Ward, 1982) so as to be expressed constitutively at low level from the int promoter in the lysogen BL21(DE2) or by IPTG induction of the lacUV5 promoter in the lysogen BL21(DE3) (Studier & Moffatt, 1986). The T7 deletion mutant 4107 removes 4419 bp of T7 DNA (Table 1), eliminating the entire coding sequences for T7 genes 0.5-1, and is completely unable to form plaques unless T7 RNA polymerase is supplied by the host (Studier & Moffatt, 1986). 4107 DNA has no sequence homology with the cloned fragment containing the gene for T7 RNA polymerase in plasmid pAR1219 and therefore viable phage particles cannot be generated by homologous genetic recombination between them. DCAT4 is a derivative of D69 in which the chloramphenicol acetyl transferase gene under control of the f10 promoter and Tf terminator for T7 RNA polymerase was inserted into the BamHI site in the orientation to be transcribed from the int promoter (constructed by J. J. Dunn of this department). The lysogen BL21(DCAT4) is only slightly more resistant to chloramphenicol than is BL21 itself, but addition of plasmid pAR1219 greatly increases resistance because of transcription by the basal level of T7 RNA polymerase from the uninduced plasmid. Induction of pAR1219 interferes with the growth of BL21(DCAT4), but chloramphenicol resistance can still be measured by spot test or streaking. Plasmids pLysS and pLysE carry the gene for T7 lysozyme in the BamHI site of pACYC184 in the silent or expressed orientation (Studier, 1991). Plasmid pAR2197 contains the coding sequence for b-galactosidase under control of the f10 promoter for T7 RNA polymerase and the gene 10 translation signals in a derivative of pBR322 (Dubendorff & Studier, 1991).

Screening for in vivo transcription activity Two tests were used to screen for transcription activity by the mutant polymerases in vivo. The first was the ability to inhibit the growth of a derivative of phage l that carries the f10 promoter for T7 RNA polymerase inserted into the BamHI site of D69. The f10 promoter directs transcription leftward relative to the genetic map of l in phage strain lf10L and rightward in lf10R. Both strains form plaques normally unless the host supplies T7 RNA polymerase. The level of T7 RNA polymerase supplied by induced pAR1219 completely inhibits plaque formation by both phages, and uninduced pAR1219 allows lf10L and lf10R to form small and medium plaques, respectively. The initial host for the mutant plasmids, HB101, is not an ideal host for plating l phage, and plasmids were usually transferred to a host such as ED8739 to assay the inhibition. The second assay was for the ability of the T7 RNA polymerase supplied by a mutant plasmid to confer chloramphenicol resistance to BL21(DCAT4). Cultures of BL21(DCAT4) carrying a mutant plasmid were tested by streaking or spot testing on plates containing chloramphenicol in the presence or absence of IPTG, using wild-type plasmid or no plasmid as controls.

Screening for protein synthesis Cultures carrying mutant plasmids were induced with IPTG at 37°C for four hours and cells were collected by centrifugation, resuspended in buffer containing sodium dodecyl sulfate, and placed in a boiling water bath for three minutes. Samples equivalent to 10 ml of original culture were subjected to electrophoresis through 10% to 20% (w/v) polyacrylamide gradient gels in the presence of sodium dodecyl sulfate, and the proteins were visualized by staining with Coomassie brilliant blue. Full-length polymerase protein was readily apparent, as were many different truncated mutant proteins.

JMB—MS 615 166 A set of T7 RNA polymerase deletion mutants T7 mutants with deletions affecting gene 1, the gene for T7 RNA polymerase, were isolated from a lysate of wild-type T7 grown on BL21(DE2). Clarified lysate was diluted 100-fold with tryptone broth containing 10 mM EDTA and heated at 55°C for 15 minutes to enrich for deletions (Studier, 1973). About 1000 surviving phage were used to grow a second lysate on BL21(DE2), and these phages were subjected to the same heat treatment. Survivors of either treatment were plated on BL21(DE2) and individual plaques were spot tested to identify gene 1 deletion mutants by their inability to grow on BL21. Thirty-two deletion phages were isolated from the first heated lysate (4105 to 4108 and 4125 to 4152) and 16 from the second (4109 to 4124). Approximately 10% of the survivors from the first heated lysate and 35% from the second were gene 1 deletions. The size and approximate position of each of these 48 gene 1 deletions was determined by restriction mapping. The exact location of 15 of them was determined by comparing the nucleotide sequence of the mutant and wild-type DNAs. As expected (Studier et al., 1979), each deletion arose by a crossover within a short repeated sequence in the T7 DNA (Table 1). Most of the deletions in this collection have their right end within gene 1 and their left end in the large dispensable region to the left of gene 1. The right ends are well distributed for mapping mutations within gene 1, but the few deletions we isolated that removed the right end of gene 1 were not well suited for mapping. Therefore, we constructed deletion 3490, which removes the coding sequence from amino acid residue 57 rightward past the end of the gene and into the R1.1 RNase III cleavage site, but does not affect any genes to the right of gene 1. Two cloned fragments of T7 DNA (bp 3143 to 3342 and 5881 to 6119) were joined in a plasmid through a 10 bp linker containing a BamHI site, and the deletion between the cloned fragments was introduced into wild-type T7 by homologous recombination during infection. The deletion mutant was isolated by plating the lysate on BL21(DE2) and spot testing individual plaques for inability to grow on BL21. The position of the deletion was confirmed by restriction analysis. Mapping of lethal T7 RNA polymerase mutations in the cloned gene Lethal mutations were mapped by three-factor genetic crosses between the cloned gene, deletion 3490, and an overlapping deletion that removes the left end of gene 1 (Figure 1). Crosses were made in HMS174(DE2) or BL21(DE2) to supply active T7 RNA polymerase needed for transcribing the T7 genes required for recombination. Overnight cultures were diluted 20 to 100-fold into M9TB and grown to A600 of about 0.2 (5 × 107/ml). Samples of culture were mixed with an equal volume of a mixture of the two deletion mutants (each at a concentration of 5 × 108/ml), further incubated at 37°C for an hour to allow growth and lysis, and 5 ml of the resulting lysate was plated on BL21. Recombination with the chromosomal copy of the polymerase gene usually contributed a background of 100 to 300 plaques. Recombination with the mutant plasmid can increase this background of viable phage only if the lethal mutation lies outside the region of overlap between the two deletions, since any mutation within the region of overlap must be acquired by any recombinant phage that acquires the polymerase gene (Figure 1). In general, negative and positive results were easily distinguishable,

Novel Mutants of T7 RNA Polymerase

with at least 1000 plaques per plate when viable recombinants could be produced, and usually complete clearing of the lawn. In a few cases, the mutation was not completely lethal and phages that carried it could form tiny plaques. In such cases, the result could usually be obtained by examining the plates early, before the background of tiny plaques made it difficult to see the larger wild-type plaques. Placing mutant T7 RNA polymerase genes in the chromosome A recombination and selection procedure was used to transfer the T7 RNA polymerase gene under control of the lacUV5 promoter from mutant derivatives of plasmid pAR1219 into the BamHI site of the lD69 cloning vector. The recipient for recombination was l3680-L or l3680-R, which were created by inserting into the BamHI site of D69 (in the leftward or rightward orientation) the appropriate BamHI-HindIII fragment from plasmid pAR3680, which is exactly equivalent to pAR1219 except that the fragment under control of the lacUV5 promoter contains T7 gene 2 (bp 8766 to 9277) instead of T7 gene 1. (The lacUV5 fragment also contains the lacI gene to supply lac repressor.) These recipient phages can replace their copy of gene 2 with gene 1 from pAR1219 or its mutant derivatives by genetic recombination with the homologous flanking sequences during infection. T7 gene 2 is an inhibitor of E. coli RNA polymerase that interferes with l growth if expressed during infection. Both recipient phages grow normally in the absence of IPTG, indicating that expression from the uninduced lacUV5 promoter is low enough not to interfere. However, when plated in the presence of IPTG, l3680-L forms no plaques and l3680-R forms only small plaques, whereas the same phages containing gene 1 instead of gene 2 form normal plaques in the presence of IPTG (if the host cell contains no promoter for T7 RNA polymerase). Therefore, gene 1 may be transferred from plasmid to phage simply by growing one of the recipient phages on ED8739 carrying the pAR1219 derivative, plating the progeny on ED8739 in the presence of IPTG, and selecting a normal plaque. Restriction analysis of the phage DNA confirms the presence of gene 1, which can then be inserted into the chromosome by forming a lysogen. The resulting lysogens are equivalent to lysogens of DE3 or DE4 except that they contain the small BamHI-HindIII fragment from pBR322 downstream of gene 1. DNA sequencing DNA sequencing used Sequenase (US Biochemicals) and deoxyadenosine 5'-[a- 35S]thiotriphosphate according to the supplier’s instructions, priming on purified plasmid or phage DNA with synthetic oligonucleotide primers. Sequencing patterns for X134 are shown in Figure 2 of Studier (1989).

Acknowledgements We thank John Dunn for permission to cite his unpublished work on T7 RNA polymerase mutations that prevent DNA maturation and packaging. This work was supported by National Institutes of Health grant GM21872 and by the Office of Health and Environmental Research of the United States Department of Energy.

JMB—MS 615 Novel Mutants of T7 RNA Polymerase

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Edited by M. Gottesman (Received 27 February 1995; accepted 18 April 1995)