transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites ...
Nucleic Acids Research, Vol. 18, No. 20 5961
k.) 1990 Oxford University Press
Selection for mutations in the PR promoter of bacteriophage lambda Susan Brown1, Julia Ferm1, Scott Woody1' 2 and Gary Gussin1' 2 1Department of Biology and 2Genetics PhD Program, University of
Iowa, Iowa City,
IA 52242, USA
Received July 31, 1990; Revised and Accepted September 14, 1990
ABSTRACT Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+ ' strand as template) toward the M13 origin of replication (orn). Lethality can be relieved by mutation of PR, repression of the promoter by the X cl repressor, or by insertion of a strong transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR. This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively. INTRODUCTION Cells harboring high-level expression plasmids frequently must be maintained under conditions in which expression of a cloned gene is repressed, usually because the expressed protein is toxic to the host cell in high concentrations. In some cases, excess transcription per se has been shown to cause plasmid instability or reduce copy number, presumably by interfering with plasmid DNA replication (1, 2). We have taken advantage of a similar effect on multiplication of phage M 13 to isolate mutations in the phage X promoter, PRWe have found that insertion of a DNA fragment containing PR is lethal to M13 vectors when transcription proceeds (counterclockwise in Fig. lb) toward the M13 replication origin (ori). By selecting phage able to form plaques in spite of the presence of PR, we identified 14 different point mutations at 8 consensus sites in the -10 and -35 regions of the promoter. Mutations at two sites (-9 and -10; for numbering of sites, see Table 2) were not found, but were subsequently introduced using oligonucleotide-directed mutagenesis (3). Phage containing mutations at these sites formed plaques, but the effects of the mutations on transcription from PR in vitro were not so severe as the effects of mutations that arose spontaneously. Lethality could also be relieved by providing X cI gene product (repressor) to block transcription initiation from PR or by insertion of transcription terminators between PR and M13 ori. However, in one case the inserted terminator did not prevent the accumulation of PR- mutations when phage were propagated in the absence of repressor.
Selection for survival of an M13 recombinant phage has also been used to generate a mutation in the X promoter, PRM, and in principle could be used to isolate mutations in other strong promoters controlled by trans-acting regulatory proteins.
MATERIALS AND METHODS Bacteria and plasmids E. coli strain JM103Y is a derivative of JM103 (4, 5), which was cured of phage P1 in this laboratory. Strains CJ236 and MV1190, the dut-ung- and dut+ung+ strains available from BioRad Labs. (Richmond, CA), were used for oligonucleotidedirected mutagenesis. The Su- strain CSH50 (6) was mated with JM109 (5) in the laboratory of M. Feiss to obtain CSH50/F'lac. Plasmids: pXY312-1, provided by J. Roberts, contains the late transcription terminator (tR') of phage 82 and was derived in the Roberts laboratory from pXY312 (7); pMS316 contains the transcription terminator tt of phage P22 (8); pMS317 (9) produces repressor under the direction of the X PRM promoter. [pMS316 and pMS317 were supplied by M. Susskind.]
Recombinant phages As outlined in Fig. 1, PR/PRM-containing DNA fragments (Fig. la) were inserted into M13 mp8, mp9, mpl8, or mpl9 (5). In some cases (see RESULTS) recombinant phages were propagated in the presence of repressor supplied by a prophage, Ximm434gt2 [lacPuv5-cI], an imm434 derivative of Xgt2 (10), containing the lacPuv5-cI fusion from pKB280 (11). Growth of phage stocks, efficiency of plating measurements, and phage competition experiments Phage stocks were grown from single plaques for 5-6 hrs at 37°C according to standard procedures (4). To measure plating efficiencies, log phase cells (about 5 x 108/ml) were centrifuged at room temperature and resuspended in one-tenth volume of ML medium (12). Phage were incubated with 0.1 ml of the concentrated bacteria for 5 min at 37°C and plated using a standard plaque assay. To isolate mutants, phage were plated without pre-incubation in a mixture containing 0.2 ml of log phase cells (ca. 5 X 108/ml). Competition experiments were performed by infection of exponentially growing JM103Y (about 108/ml) at an approximate moi of 3-4 of each phage type;
5962 Nucleic Acids Research, Vol. 18, No. 20 cultures were diluted 1:4 every 90 min. After 4.5 hr at 37°C progeny phage were assayed on JM103Y (Su+) and CSH5O/F'lac (Su-).
Enzymes and chemicals Commercial enzyme sources: restriction enzymes, New England Biolabs (Beverly, MA) or Promega Corp. (Madison, WI); DNA polymerase I (Klenow fiagment), New England Biolabs; T4 DNA ligase, Bethesda Research Laboratories (Gaithersburg, MD). E. coli RNA polymerase (RNAP) was purified by J.-J. Hwang (12). Purified repressor was provided by R. T. Sauer. Runoff transcription assays In vitro transcription was assayed as described previously (12) except that RNA polymerase (50-60 nM) was incubated with template DNA (2 nM) for only one min prior to addition of nucleoside triphosphates and heparin. The DNA template was the 678-bp EcoR;IHindIll fragment shown in Fig. la. x-[32P]-UTP was obtained from Amersham-Searle (Chicago, IL). Autoradiograms were scanned with a BioRad Video Densitometer.
Oligonucleotide-directed mutagenesis of PR Mutagenesis of PR was directed by a 21-base primer complementary to the X 1 strand. The primer was synthesized on an Applied Biosystems 340S DNA synthesizer with all four nucleotides incorporated in equal proportions at sites corresponding to positions -9 and -10 in PR. This primer was hybridized to single-stranded M700 DNA prepared following infection of the dut-ung- strain CJ236 as outlined in the Mutagene kit (BioRad Labs., Richmond, CA). DNA sequence analysis DNA sequences were determined by the dideoxy chain terminating method (13), using 'Sequenase' (US Biochem., Inc., Cleveland, OH). Primers were (i) a commercially available primer (New England Biolabs, Beverly, MA) hybridizing to lacZ; (ii) a 29-base primer complementary to the r strand of the X cI gene and extending (5' to 3') from bp 37,819 to bp 37,847, which was synthesized on a Beckman System 1 DNA synthesizer.
RESULTS Lethality of PR-directed transcription counterclockwise toward od To facilitate oligonucleotide-directed mutagenesis of the X PRM promoter, we attempted to insert a PRm-containing DNA fragment into M13mp8 (4). In the desired orientation, transcription of the XcI-lacZ fusion would be under control of PRm (Fig. Id). However, the recombinant phage (M200) formed plaques only in the presence of repressor, which could be supplied by a X prophage or by the multicopy plasmid pMS317 (9). In the absence of repressor, the efficiency of plating (eop) of M200 phage stocks (prepared in the presence of repressor) was approximately i0-4 to 10-5 (Table 1, line 3). The probable cause of lethality in the absence of repressor is that the lytic promoter, PR, which is closely-linked to PRM and directs transcription in the opposite direction, is sabotaging M13 replication (1, 2). Even the amount of repressor provided by a prophage (14, 15) apparently reduces transcription from PR sufficiently to allow phage M200 to form normal plaques.
Lethality is unlikely to be due to overproduction of a protein because phage MIOl, which contains a similar fragment in reverse orientation, plates with equal efficiency in the presence and absence of repressor (Table 1, line 2).
Isolation of mutations in PR M200 was plated on the non-lysogenic bacterial strain JM103Y to analyze the survivors by DNA sequence analysis. Of the first 17 survivors analyzed, all contained single base pair changes in the -35 and -10 consensus regions of PR. Although no precautions were taken to insure the isolation of independent mutants, there were 6 different mutations at 5 different sites. Subsequently, a new stock of the same phage (M200) was plated on JM103Y and 33 additional survivors were analyzed. In this case, 28 of the isolates contained point mutations in consensus nucleotides in PR, and 5 contained rearrangements and/or deletions. There were 7 different mutations at 5 different sites, but one mutation (G:C to A:T at -33) appeared 17 times, most likely because it arose early in the growth of the phage stock used in the isolation of survivors. Sequence changes identified in this way are listed in Table 2 (line 2). Similar experiments were performed with strain M500, which differs from M200 in that it contains the up promoter mutation prmup-1 in PRM and the UAG nonsense mutation am34 in cI (16). In these experiments, we were surprised to find that the efficiency of plating of M500 in the absence of repressor (strain JM103Y) improved by 2-3 orders of magnitude during repeated propagation of the phage in the presence of repressor [strain JM103Y(pMS317)]. Among the mutations that contributed to the higher eop on JM103Y, 10 were PR- mutations'. These included 7 different mutations of 6 different consensus nucleotide pairs. The distribution of mutant sites (Table 2, line 3) does not appear to be significantly different from that obtained with strain M200 in which PRM was wild-type. Experiments with phage M500 also yielded 4 mutants with deletions or rearrangements.
Effects of transcription termination signals We next attempted to create viable recombinants by placing a strong transcription terminator between ori and PR' Recombinants containing both tant, the ant terminator of phage P22 (8), and t82, the terminator tR' of phage 82 (7), formed somewhat smaller plaques in the absence of repressor, but their eop's were only slightly lower in the absence than in the presence of repressor (Table 1, lines 4-8). However, after oligonucleotide-directed mutagenesis of PRM in constructs containing t82, all the isolates examinedifor the desired mutation (a deletion of the nucleotide pair at position -34 with respect to P,m) contained point mutations in PR. Among 23 isolates examined (Table 2, line 4), we found 7 mutations at 7 different sites; one mutation, a change of a T:A pair to a C:G pair at -35 l Some of the mutations that led to improved plating efficiency appear to have arisen at other, unidentified sites in the phage genome. We cannot explain the unusual behavior of this phage based on known properties of thepnnup-l mutation. However, these data suggest that in some circumstances, inhibition of M13 replication can be relieved by mutations that may not directly affect transcription initiation from PR. In addition to these complications, the clam34 mutation reverted at high frequency to cI+ in strains containing pMS317, a multicopy plasmid encoding the cI gene; most likely this was due to marker rescue. Reversion of the cI mutation resulted in a change in plaque color in the presence of X-gal from pale blue to dark blue in spite of the fact that JM103Y is Su+. 7his indicates that the cIam34-lacZae fusion protein is not produced in excess under these conditions.
Nucleic Acids Research, Vol. 18, No. 20 5963 PR ¢PRM
a. cro'
If rz
Z=> -~
c.
,
RI1
CC
ac
4l~I'lPR
IacZa
PWRM
c/'
ori
cl'
o
Mio1 RI1
H
a
P~~~~~~~ P
n
R
ori
11
t
PRM
lacZa
ci'
d.
H
RI'
M200, M400, M500
jIJ b.
lacZoc
ori lacP
ori
1R
PR
PM
lacZ a
cl'
M1401
e.
H
RI RI Bm/ I/I
Bg
v
polylinker
VI lx fill ~~~~~~~T
_
VoVl
ori f.
111
tant RInBm RI, Bm
R
m
ci' lacZa .'
RM
R I"
M1501
H
Figure 1. Structure of recombinant phages. (a) Lambda DNA fragments obtained from the region shown were inserted into various M 13 vectors (b). The designation EcoRIt denotes sites created by ligation of decameric EcoRI linkers (Collaborative Research, Waltham, MA) to the 890-bp HaeUI fragment extending from bp 37,248 to bp 38,137, or to the site obtained by modification of the HaellI site in cro as outlined by Maurer et al. (15). (b) M13 vectors. Roman numerals denote M13 genes; T is a strong transcription terminator (23). (c) In MIOI, the EcoRI fragment shown in (a) was inserted at the EcoRI site of M13mp8. To produce an inframe PR-cro'-lacZa fusion, the polylinker was cut with AccI to generate 2-bp single-stranded ends, which were filled in with the Klenow fragment of DNA polymerase I (PolI-K), and religated. (d) In M200, DNA extending from the EcoRI site (38,137) to HindIl (37,459) was inserted between the EcoRI and HindI sites of M13mp8. PRM directs the synthesis of an in-frame cI-lacZa fusion peptide. M400 contains the same HindIll/EcoRl# insert as M200, but the fragment was isolated from X1 12 [cI+-PRM+-cro'] (15) and ligated to EcoRI/HindlHI-cut M13mpl8 RF. [The EcoRI site in phage X1 12 was created by altering the HaeIII site at bp 38,137 (see 15); thus, M200 has an additional 2 bp at the EcoRI# site.] M500 contains the analogous HindLII/EcoRI # fragment from XI 12 [cIam34-prmup-l-PR-cro'] (15) inserted into Ml3mp8. In M500, the mutant prmup-l promoter directs the synthesis of an in-frame cI-lacZ&t fusion peptide. M700 (not shown) contains the same insert as M400, but in the opposite orientation (in M13mpl9). (e) M1401 contains (from left to right): A 98-bp EcoRI fragment containing t82 (tR' from phage 82) (purified from pXY312-1); a 644-bp HindllBglII fragment (see a) inserted between the BamHI and HindlII sites of M13mpl8. (f) M1501 contains (from left to right): polylinker DNA extending from the EcoRI to the BamHI site of M13mpl8; a 266-bp BamHIlEcoRl fragment containing tt,,, a P22 transcription terminator isolated from pMS316; the EcoRIP#HindlIl fragment from X1 12 [cI+,PRM+] DNA (see M200). The polylinker EcoRI site (RI') was destroyed by cleaving with EcoRI, filling in single-stranded ends with Poll-K, and religating. Restriction sites: EcoRI (RI); BamHI (Bm); Bgll (Bg); HindIII (H). Details of constructions are available on request.
Table 1. Efficiencies of plating of M13 derivatives
pR 1. M008 2. MIO 3. M200 M400 4. M1400 5. M1401 6. M018 7. M1500 8. .M1501
Vector
orientation
mp8 " " mpl8 "
PR -lacZ PR -on PR -on
no insert
not present
PR -Oni mpl8 " "
no insert not present
PR-On
terminatora none none none none t tant none t82 t82
Infective Center Assays eopb plaque size +++ +++
++ ++
+++ ++ + ++ ++
++
1.09 1.05 app. 10-5 app. 10-5 1.11 0.95 1.01 1.01 0.90
tant is the transcription terminator for the ant gene of phage P22; t82 is the late terminator tR' of phage 82. In M1400 and M1500, the inserted lambda fragments of M1401 and M1501 (Fig. 1) are deleted. b Number of plaques on JM103Y divided by number on JM103Y(X); results are averages of four or more experiments.
a
(with respect to PR) was present in 14 of the isolates. Thus, the terminator was insufficient by itself to prevent PR from inhibiting propagation of an M13 vector containing PR directed toward ori. On the other hand, a phage containing tant, which exhibited essentially the same plating characteristics as the phage containing t82, did not accumulate mutations in PR when propagated in the absence of repressor: 8 out of 8 single-plaque isolates
were
PR+.
Effects of mutations on activity of PR Based on established consensus sequences for the -35 and -10 regions of E. coli promoters (Table 2), all the point mutations isolated should have been PR- mutations. We confirmed this conclusion for four of the mutations isolated in a cI+PRm background (Table 2, line 2). In run-off transcription assays (Fig. 2, lanes 3-6), levels of transcription were reduced by at least 85-90% for each of the four mutants tested.
5964 Nucleic Acids Research, Vol. 18, No. 20 Table 2. Nucleotide sequence changes Of PR mutants wild-type and mutant sequencesa -35 region 1. Wild-type PR 2. M200 PR(mp8 [wt]) 3. M500 PR(mp8 [pnnup-1]) 4. M1401 PR(mpl8 [1t82])b 5. Summary of PR- mutations
-35 T C
-10 region
-30 T
A
G AC
A G
C AG
A
G
AT
C
C
A
AC
C
AC
G
-12 g A GC
t.
T
A
A
-7 T C
G
C
A
G
T
A
AGT
GC
GT
CA
a In the wild-type sequence, lower case letters denote nucleotides that do not agree with consensus sequences (26); each line of the table presents nucleotide changes associated with single base pair substitutions isolated as described in the text. Nucleotides are numbered relative to the PR transcription startsite. b Since these mutations were isolated as part of an attempt to mutagenize PRM, some of the PR - mutant isolates (-35C, -31A, -1 IG, -7A) contain a deletion of the base pair at position -34 with respect to PRM and some (-35C, -34C, -33A, -31A, -8T) do not.
Table 3. Phenotypes of PR mutants inf. center assays Vector
PR allelea
plaque size
eopb
% Transcriptionc
M13mpl8
wild-type -OC I-OG -OA -9C -9C/1OA no insert
-
< .004 1.04 1.11
100 57 1:28 52 ± 17 45 ±13 30 ±17 5.6 ±1.9 not tested
-11G -7C -32G -33A
+++ +++ +++ ++
M13mp8
+ ++ ++ +++ +++ +++
1.16 1.07 1.01 1.05 1.11
1.9±2.0
1.02 1.01
4.6± 3.4 8.345.1 11.8±+ 3.8
1.08
a Numeral indicates position relative to PR transcription startsite; letter indicates mutant nucleotide in X 1 strand. b Number of plaques on JM103Y divided by number on JM103Y(X). c Intensity of band in autoradiogram relative to that of wild-type band determined densitometrically; results are means (± S.D.) of four experiments similar to that summarized in Fig. 2, performed at 60 nM RNAP.
Oligonucleotide-induced mutations The mutations listed in Table 2 occurred in 8 of 10 sites that match the consensus sequence in the -35 and -10 regions of PR. Conceivably, mutations at the remaining sites do not reduce transcription in vivo sufficiently to permit survival of phages in which PR directs transcription toward oni. To investigate this possibility, we used oligonucleotide-directed mutagenesis with an ambiguous primer (see MATERIALS AND METHODS) to induce mutations at positions -9 and -10 in PR. Five mutations (listed in Table 3) were isolated in phage M700, a derivative of M13mpl9 in which PR directs transcription clockwise toward lacZoa; this avoids problems associated with a potential reduction in viability of the mutants in the absence of repressor. To test the effects of the mutations on phage viability, the HindIllEcoRI 678-bp fragments (see Fig. la) containing the mutations were isolated from M 13mp19 and inserted (in opposite orientation) into M13mpI8. The mpl8 derivatives were propagated in the presence of repressor supplied by the prophage, Ximm434gt2 [lacPuv5-cI] (see MATERIALS AND METHODS). Use of this prophage guards against 'reversion'
of mutations in PR by marker rescue from a PR-containing prophage. All of the oligonucleotide-induced point mutations significantly enhanced the ability of phages containing them to form plaques in the absence of repressor (Table 3). However, there was a consistent difference in plaque size in the order (from largest to smallest): -9C, -9C/-10A > -10A, -lOG > -10C. These data indicate that the mutations introduced by oligonucleotide mutagenesis do relieve the inhibition of plaqueformation that we attribute to PR' The effects of the oligonucleotide-induced mutations on transcription initiation from PR in vitro are not so severe as the effects of mutations isolated spontaneously. These effects also correlate roughly with the effects on plaque-formation (Fig. 2, lanes 7-11; Table 3), and are comparable with the effects of mutations at positions -9 and -10 on transcription from the ant promoter of phage P22 (17; Susskind, pers. comm.). Since the mutations permit Ml 3 derivatives to form plaques in the absence of repressor, the question remains: why didn't they appear in the population of spontaneous mutations isolated by plating phage stocks on JM103Y? Results of a simple
Nucleic Acids Research, Vol. 18, No. 20 5965 Table 4. Competition between PR mutants
Phage 2 mpl8(am+)
Initial Titerb (pfu/ml)
Final Yield1b (pfu/ml)
Input Ratioa
Output Ratioa
Expt. I (1) -35C (2) -35C (3) -33A (4) -33A
-35C -9C -33A -9C
6.1 x lO8 6.7x 108 7.5 x 108 5.7 x 108
2.0x 1012 1.6x 1012 2.3 x 1012 2.3 x 1012
1.3 1.3 0.9 0.9
5.4 1.1 4.4 0.8
Expt. II (5) -35C (6) -35C (7) -33A (8) -33A
-35C -lOC -33A -lOc
6.2x 108 5.9x 108 6.3 x 108 6.0x 108
1.6x 1012
1.2
1.3x 1012 2.7 x 1012
1.3
> 10 < 0.01 > 10 < 0.01
Phage 1 mp8(am)
1.1 1.1
1.5x 1012
a Ratio of Phage 2 to Phage 1, corrected for relative efficiency of plating of phage 2 on JM103Y and CSH/F'lac. Input ratio, 0 hr; Output ratio, 4.5 hr. b Based on total phage titers on JM103Y. Initial titer, 0 hr; final yield, 4.5 hr.
competition experiment (Table 4) demonstrate that at least two of the mutants are at a competitive disadvantage relative to two spontaneous mutants during growth of phage cultures in the absence of repressor. This was tested by mixed infection with two different M13 phages containing PR- mutations. In each experiment, one phage was derived from M13mp8, which contains an amber mutation in gene H; the other phage was derived from M13mpl8, which contains a wild-type gene II. Progeny after several cycles (4-5 hr) of phage multiplication in exponential cultures were assayed and scored by plating on an amber-suppressing host (JM103Y) or a nonsuppressing host
wt 1
Mutation in PRM To determine whether this method of selecting promoter mutations would work for some other promoter, we studied the survival of phages in which the PRM wild-type or the uppromoter mutant, prm up-1, direct transcription counterclockwise toward on. For PRM, cI gene product (repressor) acts as an activator. Wild-type PRM does not affect phage viability either in the presence or absence of repressor, nor does prm up-I have
11G 3
7C 4
32G 33A 5 6
9C 7
10C lOG 8 9
1OA 10
9C/ 1OA 11
ig L
(CSH50/F'). When the two competing phages contained the same PRmutation, competition strongly favored the am+ (gene II+) parent even though the experiment was performed in the amsuppressing host (Table 4, lines 1, 3, 5, 7). However, when the am+ parent contained the mutation - lOC in PR' the phage was unable to compete with the amber mutant parent containing either the -33A or -35C mutation (compare output ratios in Table 4, lines 6 & 8), consistent with the fact that the mutation - lOC has a much less severe effect on PR activity in vitro than -33A and causes the greatest reduction in plaque size. [Like -33A, the mutation -35C would be expected to be a strong down mutation based on its effect on the -35 region consensus sequence.] Ml3mp8 containing the mutation -9A is a more effective competitor than phage containing - lOC against both mpl8[-33A] and mpl8[-33C] (Table 4, lines 2 & 4), but is not so effective as mp8[-33A] or mp8[-33C], respectively. Although output ratios vary somewhat from experiment to experiment (compare lines 1 & 5; 3 & 7), the basic pattern is unchanged, and titers of the -lOC mutant are always < 1010/ml at 4.5 hr. Thus, the ability of a mutant to compete is positively correlated with its effect on transcription and inversely correlated with its effect on plaque size.
wt 2
.: .
..p..
..
--
R
IRR
PR
Fi1re 2. Transcription from mutant promoters in vitro. Reactions were performed under standard conditions (12) using a 678-bp HindIll/EcoRI fragment isolated from the appropriate M 13 phage (Figure Id). RNAP (50 nM) and DNA (2 nM) were incubated at 37°C for 1 min prior to addition of NTPs and heparin (50 ,ug/ml). Transcription was allowed to proceed for 15 min. For the reactions containing repressor (lane 2), repressor (final concentration, 0.27 AM) and DNA were incubated for 10 min prior to addition of RNAP. Lanes 1 and 2, PR+ DNA in absence and presence of repressor, respectively; lanes 3-6, spontaneously isolated mutants, and lanes 7-11, oligonucleotide-induced mutants, in the absence of repressor. The 115-nucleotide long, PR-specific transcript is indicated by an arrow.
an effect in the absence of repressor. However, in the presence of repressor, the up-I promoter causes M13 to make very small plaques. Large plaques appear at a frequency of about 10-4 to 10-5. Phage from two large plaques were picked, purified, and subjected to DNA sequence analysis. In both cases, these phage contained a substitution at the up-1 site, which changed the -35 consensus region from TAGACA to TAGAAA (in wild-type PRM, the sequence is TAGATA).
DISCUSSION Efficient transcription toward M 13 ori is lethal to phage multiplication, presumably because phage DNA replication is inhibited. Since PR should be a strong, repressible promoter only in double-stranded DNA, it is likely that PR-directed
5966 Nucleic Acids Research, Vol. 18, No. 20 transcnption inhibits M13 RF replication. Lethality can be used as the basis of positive selection for down promoter mutations providing the promoter is strong, and that the phage can be propagated under conditions in which the activity of the inserted promoter is reduced. Thus, PRM is lethal to M13 only when it contains an up mutation and is activated by repressor. PR is not lethal in the presence of repressor supplied by a single prophage, and lethality is substantially eliminated even by PR- mutations that reduce transcription only about 2- to 3-fold in vitro (Table 3). Transcription might interfere with normal initiation of RF replication if (i) transcription through ori prevented assembly of replication complexes or normal priming of replication initiation (18), (ii) transcription from the + strand template caused supercoiling of RF DNA (19, 20) in the opposite sense to that induced by normal transcription from M13 promoters, (iii) unwinding associated with transcription approaching sufficientdy close to (but not through) on caused structural changes in RF DNA that would inhibit initiation, (iv) the - strand RNA itself inhibited some unknown aspect of phage development in
suggests that the inhibitory effect of transcription from PR is cisspecific; however it is also possible that because of the limited number of adsorption targets (F-pili) for the phage many cells are only singly infected. It is also possible that the mutations at -9 and -10 are at an additional selective disadvantage in lysogenic cultures because they are in the operator segment OR1 and should affect repression of PR (see 25). One advantage of this type of selection for promoter mutations is that it is based on in vivo activity of the promoter. Unfortunately, the relationship between promoter strength and M13 phage replication is not easy to quantify. In particular, a reduction in phage production by as much as a factor of 10 might not alter the efficiency of plaque formation (M. Russel, pers. comm.). Finally, when cloning a strong promoter into M13 (and possibly into plasmid vectors), it is important to be aware of the possibility that mutations in the promoter may accumulate even under conditions in which plaque or colony formation are not detectably affected.
trans.
Although transcription terminators inserted between PR and ori permit PR-containing M13 phage to form plaques, accumulation of mutations in the absence of repressor in the case of t82 indicates that the terminator by itself does not completely prevent lethality. A possible explanation for this phenomenon is that although t82 is more than 90% effective in its natural context (Roberts, pers. comm.), termination is not efficient on the recombinant template. We are investigating the possibility that PR is a naturally 'antiterminating' promoter. This could explain why termination at tRl of lambda is only about 50% efficient (D. Court, pers. comm.). Sequences close to promoters have been shown to affect transcription termination efficiency in at least two other cases (21, 22). Furthermore, decreases in termination efficiency in vivo have been observed for transcription initiated at the gyrA promoter (23). Conceivably, termination is efficient, but topological effects of transcription in the region between PR and the terminator inhibit replication. This is unlikely because the different effects of tt and t82 do not correlate either with the length of transcript produced (the transcript in the tt construct is longer than that produced by the t82 construct) or the proximity of the termination site to ori (the distances are nearly the same for t82 and ttant). When PR directs transcription clockwise in M13, we detect no lethal effect on phage replication. This is likely to be due to the presence of the very strong transcription terminator located between genes VIII and III in M13 (24; see Fig. lb). However, it is possible that replication is inhibited only when the + strand is template for transcription. Certain of the oligonucleotide-induced mutations in PR reduced transcription from PR in vitro by only a factor of two or three. These mutations permitted formation of plaques on a non-lysogen with normal plating efficiency. However, smaller plaque sizes for these mutants suggested that they would be less frequent in the phage population than the mutants isolated spontaneously. Results of competition experiments (Table 4) agree with this interpretation. It should be noted that competition was assayed in a strain lacking repressor, while spontaneous mutants were isolated after propagation of phage in the presence of repressor. However, even in a lysogen not all M13 DNA molecules may be repressed, and thus the competition we observed could still occur. The fact that competition can be observed in cells infected at a fairly high multiplicity (about 4)
ACKNOWLEDGEMENTS We thank Dr. M. Russel for helpful discussions, Drs. M. Susskind and J. Roberts for providing plasmids, Dr. J.-J. Hwang for RNA polymerase and Dr. R. Sauer for repressor. Several of the mutants listed in Table 2, line 2 were isolated and
characterized by Raymond Fong. This research was supported by NIH grant AI17508 to G. G.; S. W. was an NIH predoctoral trainee.
REFERENCES 1. Rosenberg, M., Ho, Y.-S. and Shatzman, A. (1983). In Wu, R., Grossman, L. and Moldave, K., (eds.) Meth. in Enzymol. 101:123-138. 2. Deuschle, U., Kammerer, W., Gentz, R. and Bujard, H. (1986). EMBO J. 5:2987-2994. 3. Zoller, M. J. and Smith, M. (1981). Nucleic Acids Res. 10:6487-6500. 4. Messing, J., Crea, R. and Seeburg, P. H. (1981). Nucleic Acids Res. 9:309-321. 5. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985). Gene 33:103-119. 6. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 7. Yang, X., Hart, C. M., Grayhack, E. and Roberts, J. W. (1987). Genes Develop. 1:217-226. 8. Berget, P. B., Poteete, A. R. and Sauer, R. T. (1983). J. Mol. Biol. 164:561-572. 9. Wu, T., Liao, S.-M., McClure, W. R. and Susskind, M. M. (1987). Genes Develop. 1:204-212. 10. Panasenko, S. M., Cameron, J. R., Davis, R. W. and Lehnan, I. R. (1977). Science 196:188-189. 11. Backman, K. and Ptashne, M. (1978). Cell 13:65-71. 12. Hwang, J.-J., Brown, S. and Gussin, G. N. (1988). J. Mol. Biol. 200:695-708. 13. Sanger, F., Nicklen, S. and Coulson, A. (1977). Proc. Natl. Acad. Sci. USA 74:5463-5476. 14. Reichardt, L. and Kaiser, A. D. (1971). Proc. Naid. Acad. Sci. USA 68:2185-2189. 15. Maurer, R., Meyer, B. J. and Ptashne, M. (1980). J. Mol. Biol. 139:147-161. 16. Meyer, B. J. Maurer, R. and Ptashne, M. (1980). J. Mol. Biol. 139:163-194. 17. Younderian, P., Bouvier, S. and Susskind, M. M. (1982). Cell 30:843-853. 18. Komberg, A. (1988). J. Biol. Chem. 263:1-4. 19. Pruss, G. J. and Drlica, K. (1989). Cell 56:521-523. 20. Tsao, Y.-P., Wu, H.-Y. and Liu, L. F. (1989). Cell 56:111-118. 21. Goliger, J. A., Yang, X., Guo, H.-G. and Roberts, J. W. (1989). J. Mol. Biol. 205:331-341. 22. Telesnitsky, A. P. W. and Chamberlin, M. J. (1989). J. Mol. Biol. 205:315-330.
Nucleic Acids Research, Vol. 18, No. 20 5967 23. Carty, M. and Menzel, R. (1989). Proc. Natl. Acad. Sci. USA 86:8882-8886. 24. Schaller, H., Beck, E. and Takanamni, M. (1978). In Denhardt, D. T., Dressler, D. and Ray, D. S. (eds.) The single-stranded DNA phages. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., pp. 139-164. 25. Gussin, G. N., Johnson, A., Pabo, C. and Sauer, R. (1983). In Hendrix, R. W., Roberts, J. W., Stahl, F. W. and Weisberg, R. A. (eds.) Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. pp. 93- 122. 26. Harley, C. B. and Reynolds, R. P. (1987). Nucleic Acids Res. 15:2343-2361.
