λ nutR mutations convert HK022 nun protein from a transcription termination factor to a suppressor of termination

J. Mol. BioZ. (1990) 212, 635-643

k nutR Mutations Convert HK022 Nun Protein from a Transcription Termination Factor to a Suppressor of Termination R. Robledo, M. E. GottesmanjInstitute of Cancer Research Columbia University College of Physicians & Surgeons 701 West 168th Street, New York, NY 10032, U.S.A.

and R. A. Weisberg Section on Microbial Genetics Laboratory of Molecular Genetics Building 6, Room 306 NICHHD, NIH, Bethesda, MD 20892, U.S.A. (Received

14 April

1989; accepted 13 September 1989)

The Nun protein of the lambdoid phage HK022 blocks 1 growth by terminating transcription at (or near) the A nut sites. An HK022 lysogen carrying a fusion of the L pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two &s-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxAS. The second, boxAl (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rhodependent 1 terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to dist’al terminators. The 1 N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the borA5 fusion, but displays persistent antitermination activity in the boxA fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nw,sA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

1. Introduction Gene expression in coliphage I is temporally regulated by a system of transcription termination and antitermination (for reviews, see Friedman & Gottesman, 1983; Roberts, 1988). Following infection, transcription initiates from two divergent 2 promoters, pL and pR. Full expression of the pL and pR operon genes requires the 2 N gene product. f Author

to whom

0022-2836/90/080635U9

reprints $03.00/O

should

be addressed.

635

N stimulates transcription by forming a complex with RNA polymerase and several host proteins (Nus) that is resistant to termination (Barik et al., 1987; Horwitz et al., 1987). N-mediated antitermination is transcript-specific, since N attaches to RNA polymerase only at the I nutL and nutR sequences. The nut sites can be divided into at least two distinct regions, boxA and boxB; mutations in either inhibit the N antitermination reaction (Salstrom & Szybalski, 1978; Olson et al., 1984). The boxB region displays hyphenated dyad symmetry, 0 1990 Academic Press Limited

636

It. Robledo et al

and varies among the different members of the lambdoid phage family. The boxA sequence, on the other hand, is relatively conserved among the lamboid phage. A boxA sequence is found in the Escherichia coli rm operons, where, in concert with one or more of the host Nus proteins, it suppresses termination (Aksoy et al., 1984; Sharrock et al., 1985; Morgan, 1986; C. Squires & C. Squires, personal communication). These considerations have led to the idea that boxA is the recognition site of the host Nus proteins, and boxB that of the phage-specific N proteins. One member of the lambdoid family, phage, HK022, prevents the growth of phage L (Robert et al., 1987). The responsible HK022 function is Nun, which terminates I transcription prematurely at or near the i nut sites. Nun termination mimics N antitermination; Nun recognizes the 1 nut sequences and requires the host Nus proteins. Mutations in the boxB sequence of nutL that block N antitermination also reduce Nun termination. In this article, we report the isolation of mutations in the L nutR site that prevent Nun termination. The two mutations, both in boxA, display unexpected properties: (1) they increase the efficiency of tR1; and (2) they invert the activity of Nun, which now suppresses transcription termination at tR1. As a consequence, phage carrying a boxA mutation grow in the presence of Nun, but not in its absence.

2. Materials and Methods (a) Bacteriological Standard

bacteriological

techniques

techniques,

e.g. transforma-

tion, Pl transduction, media preparation, are as described (Silhavy et al., 1984). Selection of Tet” derivatives from a predominant population of Tet’ colonies was performed on plates containing fusaric acid and chlortetracycline (Maloy & Nunn, 1981). (b) Bacteria N99 is a gaEK.2 strR strain. Strain 6D7 is derived from strain SA500 (F- sup” his ilv stp reZA1). It is lysogenic for i, ~1857 Nam7,53 and carries a pR-cro-nutR-tRl-gal fusion (Dambly-Chaudiere et al., 1983). RW1929 is a lysogen of 6D7 carrying HK022 and HK022: :TnlO-I; it was constructed by infection of 6D7 with the indicated HK022 phage. N6559 is a recA supF strain used to select phage recombinant5 bearing the pR-gal fusion of 6D7 and its derivatives. The nu.sAtsll : TnlO mutant was obtained from D. Court. nusA1, nusB5 and nusE71 strains were gifts from D. Friedman. N7001 is LE392 (Silhavy et al., 1984) bearing the nun+ plasmid pJR177. (c) Bacteriophage HK022 was obtained by R. Hendrix. HK022 : : TnlO-I was as described (Robert et al., 1987). B187 is I imm21 clts nin5 gal8 bioll; it is used to rescue the pR-gal fusions from 6D7 and its derivatives. Y328 and Y333 are 2 ~1857 gal8 derivatives that carry the gaZEoc95 and galEocB4 mutations, respectively. Y447 is 1 ~1857 bioT76 nin3; the bio substitution extends into the N gene

and the nin3 deletion removes a terminat,or(s) between A genes P and &, permitting Y447 to propagate in the absence of N. Y1242 and Y1248 are nutR boxA and nuti? boxA derivatives, respectively, of Y447. They were constructed by growing Y447 lytically on the boxA or boxA derivatives of 6D7 (RW1984 and RW1980, respectively), and plating the lysate on the nun’ strain, N7001, at 37°C. Plaque-forming phage appeared at a frequency of 4.8 x 10e6 and 1.5 x lo-‘. respectively; the vast majority of these phage propagated only on strains providing Nun or N. Y447 not grown on OozA mutant strains plated on N7001 with an efficiency of 3.6 x 1V’: these plaque-formers grew in the absence of Nun or N. B446, i imm21 b515 b519 att24 and B141. 1 timm21 cIts biol0, were used to construct Y1259 (see sec%ion (i), below). (d) Plasmids pKK9-4 is a pBR322 derivative containing no AvaI sites; it, was used for the cloning and sequencing (see section (l), below) of the pR-gal fusions and was kindly provided by J. Brosius (Brosius, 1984). pNAS200 is a lowcopy-number plasmid carrying a pZac-N fusion (Schauer et al., 1987). pJR177, an amps tep pBR322 derivative, carries nun and other genes of HK022 on a PstI fragment (gift from J. Robert). Plasmid pJO210 is a pBR322 derivative carrying an expressed nun insert (gift from J. Oberto).

(e) Analysis

of HK022 prophage

To verify the phenotype of the resident HK022 prophage Gal+ revertant cultures were centrifuged at 4000 revs/min. The supernatant, containing spontaneously released HK022 phage, was then spotted on strain 6D7; from the center of the spot, colonies were streaked on MacConkey-galactose plates at 42°C and putative lysogens were identified by their Galphenotype. (f) Test for Cro function To test for Cro activity in the Gal+ revertants, colonies grown at 42 “C were picked to MacConkey-galactose plates at 32°C. Cro- revertants recover immunity and score as white (Gal-) colonies. Cro’ revertants remain red; Cro blocks 1 repressor synthesis. (g) Transduction

of the pR-gal fusions

The pR-gal fusions from strains RW1929, RW1980 and RW1984 were moved into strain N99 as follows: first, TetS derivatives were selected as described in section (a), above; second, an nud : : TnlO marker was introduced, by Pl transduction; finally, the pR-gal fusions were cotransduced with the nad : : Tn70 to N99 (linkage approx. 50%) selecting for Tet’, and screening for the Gal phenotype. Similarly, the pR-gal fusions from mutants RW1980 and RW1984 were moved into strain 6D7 lysogenic for HK022. (h) Construction of gal EocpR-gal futiLsions The galEoc95 and galEocB4 polar mutations were introduced into strain 6D7 and its boxA and boxA derivatives by homologous recombination with Y328 and Y333, respectively, as described (Dambly-Chaudiere et uZ., 1983).

HK022 Nun Acts at Mutant 1 nutR (i) Construction of 1 ~I857 bioT76 nutRboxA5

nin+

Y1259, 1 ~1857 bioT76 boxA nin+, was derived from Y1242, the corresponding nin5 phage. The construction was performed in several steps. First, strain N7375 (SA500 su-pF, trp bio), which carries a I imm434 A443 prophage (Adhya et al., 1977), deleted for prophage genes 0 through attR, was lysogenized with Y1242 to yield strain N7445: SA500 (A imm434: i ~1857 bioT76 nutRboxA5 A(O-attR). N7445 forms by homologous recombination between the infecting Y 1242 and the partial 1 imm434 prophage. Second, the A imm434 prophage was cured from N7445 by superinfection with B446 to yield strain 7451: SA500 (1 ~I857 bioT76 nutRboxA5

A(O-attR).

Strain 7451 is deleted for the nin region, and recombinants between the partial I prophage and a nin+ superinfecting phage must, therefore, be nin+. Finally, N7451 was infected with B141 and the imml recombinants isolated on N7437, YMC(J. imm21 h80 att80)/pNAS200. These recombinants are 1 ~I857 bioT76 n&R boxA5nin+ (Y1259). The inclusion of the N+ plasmid, pNAS200 in the selecting host, is required; Y1259 fails to

propagate in the absence of N or Nun. (j) Calmtokinase assay Cells were grown at 32°C in LB to about 2 x lO’/ml, and induced by shifting to 42°C. Galactokinase levels were determined 1 h after induction, as described (Robert et at., 1987). Extracts of strain N99 (galK2) were used as a blank. (k) Northern blot analysis Exponentially growing cells in LB at 32°C were shifted to 42°C for 15 min to induce transcription from the pR-gal fusion. RNA was extracted and analyzed as previously described, using a 1 RNA probe (I co-ordinates 38,214 to 38,399; Robert et al., 1987). Recall that the pR-gal fusion point in strain 6D7 lies at A co-ordinate 38,345. (1) DNA sequencing Strains mutations

carrying the pR-gal fusions with boxA were superinfected with B187, and recombi-

nants carrying the chromosomal fusion were selected as Fee’ on N6559 (Dambly-Chaudiere et ai., 1983). A I.1 x IO3 base-pair Hind111 fragment, derived from cuts in CI and gaZE, was cloned into plasmid pKK9-4. Amp’ Tet” colonies were screened and the DNAs were sequenced by base-specific chemical cleavages starting from the 5’ end-labeled Am1 site (1 co-ordinate 38,214), located near the 3’ end of cro, into gaZE (Maxam & Gilbert, 1980). The boxA mutation, present in strain RW1980, was

sequenced on the bottom strand as well: the recombinant plasmid pKK9-4,

fragment.

was

carrying the 1.1 x lo3 base-pair Hind111

denatured and annealed to a 17mer (1

co-ordinates 38,329 to 38,345) complementary to the “top” strand of the tR1 region, according to the Amersham protocol. DNA was sequenced by the dideoxy method (Sanger et al., 1977), using [a-35S]dATP and deoxy-dideoxy NTPs (Amersham). Sequenase enzyme (U.S. Biochemicals) was used. The bottom strand was sequenced from 1 co-ordinate 38,321 (in the tR1 region) to 1 co-ordinate 38,191 (in cro).

Nun terminates

637 transcription

607

in the X nul region

Fusion

S/rain

Nom--cI857--pR--cro--n&i--fR/--go/Em Nun ----------

_______

+

---------),I

+

Figure 1. Effect of HK022 Nun on 1 transcription. Strain 6D7 carries a pR-gal operon fusion; the point of fusion is at 1 co-ordinate 38,345, just promoter-proximal to ~11. Transcription initiates at pR when the ~I857

repressor is inactivated at 42°C. Nun provokes transcription termination in the nutR-tR1 region.

3. Results (a) Isolation of 1 nutR mutants HK022 prophage block the growth of superinfecting 3, by terminating transcription at or near the 1 nut sites. Similarly, operon fusions that include the II nutR or nutL sites are inactivated by KH022. E. coli strain 6D7 (Dambly-Chaudiere et al., 1983) bears the operon fusion: 2 pRoR-m-o-nutR-tRl-gal and the 3,cIts857 repressor (Fig. 1). Strain 6D7 is Gal+ at 42”C, the temperature at which the A repressor is inactivated. The 1 N-product stimulates gal expression, but is not essential, since tR1 is an inefficient (approx. 50%) terminator. HK022 lysogens of 6D7 are Gal- because Nun terminates transcription in the nutR region. By selecting for Gal+ revertants of a 6D7 HK022 lysogen (RW1929), we hoped to obtain mutations defining elements required for Nun termination. To avoid nun mutations we chose a lysogen carrying at least two HK022 wu%+ prophage. Spontaneous Gal+ revertants were selected at 42 “C on MacConkey-galactose indicator plates, and variants defective in Nun termination were isolated as follows. First we verified, by appropriate gal expression was temperature shift, that controlled by I repressor, and thus derived from the 2 pR promoter. We then showed that the mutants released HK022 Nun+ phage (see Materials and Methods) and that they remained Gal+ when a new HK022 was substituted for the original prophage. Next, the Gal+ variants were tested for their ability to plate superinfecting 1 imm434, a phage that is sensitive

to Nun

inhibition.

Those

that

permitted

phage growth were demonstrated to carry mutations in host genes that block Nun activity (R. Robledo, R. A. Weisberg & M. E. Gottesman, unpublished results). The Gal+ variants that were resistant to superinfecting IEimm434, and therefore able to express the Nun+ phenotype, carry pR operon fusion mutations that render gal expression resistant to Nun termination. This was demonstrated initially by transducing the fusions from the variants into strain 6D7 lysogenic for HK022; all transductants were Gal+ (see Materials and Methods).

R. Robledo et al.

638 Table 1

Table 2

Isolation of 1 nutR mutants

Sequence of boxA mutants

Galactokinaue Strain 6D7 RW1929 RW1980 RW1984 RW1988 RW1990 RW1991

-

HK022 + + + + + +

Phenotype Gal+ GalGal + Gal+ Gal+ Gal+ Gal+

-N 3.3(kO.l) @5( kO.1) 5.9( & 0.5) 62( & 1.1) 68( &-0.4) 54( 10.7) 58( kO.5)

units

Fusion: pR---- -cm

+9 86( + 05) 0.6( + 0.2) 74( 5 0.5) 62( kO.2) 6.2( & 0.2) 52( + 0.4) 68( kO.9)

Values represent galactokinase units determined 60 min after thermal induction (see Materials $ Methods); they indicate average units from duplicate samples. Fluctuations are given in parentheses. All strains derive from 6D7 and carry fusion: pR-n&R-tR1 -gal. N was obtained from the plasmid pNAS200.

The nine independent Gal+ variants carrying fusion-linked mutations could be separated into two categories: five were Cro+ and the remainder were Cro- (see Materials and Methods). The latter, we presume, carry cro-nutR deletions or cro frameshifts that inactivate nutR (Olson et al., 1984; Zuber et al., 1987; Robert et aE., 1987); this class was not further investigated. We then measured galactokinase activity in the Cro+ mutants (Table 1). The wild-type fusion strain (6D7) expressed 3.3 units of galactokinase in the absence of N and 8.6 units when tR1 is suppressed by N. Nun severely inhibited galactokinase expression (95 unit; strain RW1929), and N did not reverse the Nun effect. Mutants RW1980, RW1984, RW1988, RW1990 and RW1991 all displayed galactokinase activities higher than the parental 6D7. Furthermore, in contrast to 6D7, there was no increase in galactokinase activity when N was supplied. This unexpected N-independence of galactokinase synthesis in the mutants suggests that tR1 no longer functions as a termination signal in these strains. We shall return to this point below.

(b) Revertants RW1980 and RW1984 carry A nutR boxA mutations The sequence of the pR operon fusions in strains RW1980 and RW1984 was determined by transferring the fusions from the chromosome to i gal8 bioll by homologous recombination (see Materials and Methods). From this recombinant phage. a hIgaZE Hind111 fragment was subcloned in plasmid pKK9-4, and subsequently sequenced (see Materials and Methods). Both fusions were shown to carry transversions in the boxA region of 1 nutR (Table 2). The mutation in strain RW1984, a G to T transversion in the second nucleotide of boxA, is identical with the boxA mutation engineered in vitro by Olson et aE. (1984). Strain RW1980 carries a C to A transversion in the fifth nucleotide; we refer to this mutation as boxA16. Both mutations affect strongly conserved boxA nucleotides.

boxA + boxA boxA

~tR1

n&R-

/ boxA

‘\

boxB

~CGCTCTTA~-CTCTCTTACGCTATTA

(c) Effects of the boxA mutations on transcription termination To see whether the boxA mutations affect gal expression in the absence of Nun, the fusions present in RW1929, RW1980 and RW1984 were transduced into a non-lysogenic strain (N99). these transductants had less gal Surprisingly, operon activity than their lysogenic parents, as judged by reduced color on MacConkey-galactose indicator plates. When an HK022 prophage was introduced into the transductants, however, both mutant fusions expressed gal at high levels. Lysogenization with HK022 nun- did not stimulate gal expression. Thus, rather than isolating mutations resistant to Nun termination, our ,nutR mutant fusions were dependent on Nun activity. Table 3 shows the galactokinase levels in transductants carrying either boxA+, boxA or boxAl pR-gal fusions. The boxA mutation reduced gal expression almost threefold; boxAl inhibited to a lesser and somewhat’ variable extent, (Table 3, column 1). Introduction of a nun plasmid clone the wild-type fusion inactivated completely (Table 3, column 2). Tn contrast, Nun stimulated gal expression in both boxA mutant’ fusions. Wild-type and mutant’ fusions were both activated by N product, although the mutants responded less well (Table 3. column 3). We can now explain why N failed to stimulate gal operon expression in HKO22 lysogens of the mutant fusions (Table 1): we suggest, that transcription termination is alwady fullysuppressed by Nun in these st,rains. We believed it likely that’ the boxA mutations decreased gal expression by increasing termination at tR1, a weak, rho-dependent terminator that, lies

Table 3 EJect of nutR boxA mutations on transcription termination Plasmid n&R boxA + boxA boxA

None

pNun

IIN

31(&@5) l%?(+o.l) 2.6( k 0.3)

O,l( *@I) 6.X( f 1.5) 5.9( f0.3)

7.6( f 0.2) Y.4( f0.1) 31( 50.1)

Values represent average galactokinase units from duplicate samples, determined as in Table 1 (see note to Table 1). The pNun and pN plasmids are, respectively, the pJ0210 and pNAS200 plasmids described in Materials and Methods.

HK022

Nun

Acts at Mutant

1 nutR

639

-2900

-

I500

-

120

Figur ‘e 2. North1 ?rn blot analysis of I transcripts made in strain 6I)T and derivatives. RNA was extracted 15 min after thermal induction electrophoresed and hybridized to a labeled i RNA probe (shown at t,he top of’ the Figur be) as describe ,d by Robe *rt et al. (1987) and in Materials and Methods. Comparable quantities of RNA were applied t,o each lane, as judged by rthidium bromide staining of the gel, and quantification of the ribosomal RiYA bands.

upstream from the L/gal fusion point. To test this hypothesis, we introduced the rho15 allele into the wild-type and box.45 fusion strains. The rho15 derivatives, whether hoxA+ or boxA5, displayed high levels of galactokinase activity (data not shown). This suggests that, box.45 increases termination at a Rho-dependent, terminator, presumably tRI. Analysis of pR operon RNA, described below, is entirely consistent with this notion. (d) Analysis

of thl> ,4 pR

transcripts rind mutant strains

in the wild-type

The transcripts in the pR fusion strains were subjected to Northern blot analysis, using a 321’-radiolabeled RNA probe complementary to the region between the AvaI site in cro to the AUG of ~11. The results of these experiments are shown in Figure 2. When a wild-type fusion was induced, we observed two RNA bands about equal in intensity (lane 1). The size of the smaller RNA, approximately 230 to 310 bases, is consistent with a transcript init’iating at pR and terminating in the tR1

region. The larger RNA (approx. 14 x IO3 basepairs) represents, we believe, transcripts reading through this region into gal, and being subsequently processed (Achord & Kennell, 1974). No hybridization to the probe was seen in the absence of thermal induction, indicating that the transcripts initiated at 1 pR (data not shown). The pattern of transcription in the boxA and boxA fusions differed from the wild-type; there was a decrease in readthrough RNA and a corresponding increase in RNA terminating in the tR1 region (Fig. 2. lanes 2 and 3, respectively). The presence of Xun blocked all readthrough RXA in the wild-type fusion; all pR transcripts now terminated in the tR1 region (Fig. 2, lane 4). In contrast, Nun stimulated readthrough in the boxA and boxA mutant fusions (Fig. 2, lanes 5 and 6, respectively). These analyses support our notion that the 60~4 mutations have two effects: (1) increased transcription termination in t,he tR1 region; and (2) conversion of Nun from a termination function to a suppressor of termination. We note the apparent large increase in pR transcripts in cells expressing Nun (Fig. 2, lane 4; see

R. Robledo et al.

640

Table 4

also Robert r,t al., 1987). We have previously suggested that, in the absence of sun, pR transcripts are destabilized by processing at some point5 downstream of nutR. Alternatively, Nun may. in fact, stimulate pR transcription by releasing transcripts before the tR1 pause site. Because of the relatively small size of the pR to tR1 region, RNA polymerases paused at tR1 could block transcription initiation at pR. (e) Nun

does not suppress termination distal sites

at promoter-

The modification of RNA polymerase that N promotes at the 1 n&R site is persistent; not only is tR1 suppressed, but terminators promoter-distal to tR1 are likewise overridden. We have created additional transcription terminators in our pR-gal fusions by introducing polar ochre mutations into the first gene of the gal operon, galE. These mutations decrease the synthesis of galactokinase, the product of the downstream galK gene. Such mutational polarity is thought to result from premature transcription termination at Rho-dependent termination sites that are located promoterdistal to the mutation, resulting in decreased expression of downstream genes. These termination sites are cryptic in the absence of an upstream polar mutation because they are suppressed by translation (Adhya & Gottesman, 1978). Nun was unable to suppress polarity; we observed no increase in galactokinase activity when we supplied Nun to two different gaZEoc fusion strains (Table 4). Thus. although Nun overrides tR1 in the boxA and boxA fusions, it cannot establish a persistent antitermination transcription complex. As reported by Dambly-Chaudiere et ~2. (1983), N suppresses the polarity of these galEoc mutations when they are distal to a wild-type boxil sequence. However, the boxA mutation blocked the ability of ;1; to suppress distal terminators. The effect of boxA on polarity suppression by N depended on the polar mutation: suppression of galEocB4 polarity was nearly complete, whereas suppression of galEoc95 polarity was reduced, but’ still significant. The difference between t,he two ochre mutations may reflect the particular cryptic terminator activated by the mutation. We conclude that’, whereas boxA mutations have little effect on N suppression of tR1, they inhibit’, to a greater 01 lesser degree, antitermination by N at, more dist’al sites. (f) The effect of the nutR boxA mutations

on

phage growth

The effects of the boxA mutation on termination in the tR1 region and on Nun activity can also be observed in phage 1. We crossed nutR boxA from the fusion into AN- nin3 (Y447) by growing this phage on the mutant strain and selecting the rare recombinants that could form plaques on a Nun+ host. The nin3 deletion removes terminators distal

Effect

of

boxA mutations

on polarity

suppwssiorr

Fusion

--.-

A

1% Plasmid

F’lasmid None

pNun

pN

0.9 0.3 0.9

0.2 1% 0.1

132 14 11.9

borA + boxA boxAl

NOW

pNun

pN

(6 I.0 0%

13.4 0.9 2.5

0% 0.7 07

Fusion A: pR-n&R-tRl-galEocB4-T-K Fusion B: pR-nutR-tRl-galEoc95-T-K Values represent galactokinase units from duplicate samples. determined as in Table 1. Fluctuations are less t,han 1 unit.

to tR1, and thus permits 1 growth in the absence of N antitermination. nin phage are sensitive to Nun, however, since premature termination at or near nutR blocks expression of the distal J 0 and 1’ genes. Phage grown on a boxA host formed plaques on a Nun+ indicator with an efficiency of about 4 x 10m6, tenfold more than phage grown on a wildtype host. These plaque-forming phage, as expected. are recombinants that carry the boxA mutation, as shown by DNA sequencing (T. Patterson & D. Court, personal communication). In Table 5. we compare the plating patterns of the boxA phage to its boxA+ counterpart, I. boxA nin3 grows only in the presence of Nun or N. Its boxA+ parent, in contrast, grows only in the absence of Nun and is indifferent to N. We assume that Nun and N allow growth of the boxA mutant by suppressing termination at tR1. A similar result was obtained when boxA was crossed into 1.IV nin3; the boxA mutant phage was dependent on N or Xun for growth (data not shown). To see if Nun or K suppresses termination at downstream ,I terminators, we examined the properties of I N boxA nin+ . which contains the strong tR2 and tR3 terminators (Leason & Friedman, 1988; Court’ & Gottesman, unpublished results). Although

Growth

qf 1” nutR

Table 5 mu,tants

is ,V or iVurL depend& Plasmid

nN nutR

nit1

+ +

nin3 +

boxA boxA

nin3 +

N0r1e

pNun

pN

-.

+ +

+

-

+

+ +,‘-

Phage structure: -nin31, cI-pR-cro-nutR-tRl-0-P-tR2-tR3-Q. Phage are plated on TB agar at 37°C on N99, N99/pNun 01 N99/pN. -, No growth; f. normal plaque size: +/-. minute plaques.

HK022 Nun Acts at Mutant 2 nutR

Table 6 Inversion of Nun activity by nus mutations borA +

boxA

nus

nunm

nun+

nun-

nun+

+ AI B5 E71

4-5 54 45 3.8

0.4 8.0 11.7 7.9

1.7 1% 2.4 1.1

6.8 8% 67 4.1

Numbers represent galactokinase units; strains were grown and tested at 42°C. The experiment was repeated twice with essentially the same results.

it was possible to construct this phage (see Materials and Methods), it grows very poorly, and only on strains supplying N. These results are consistent with those reported above; although both Nun and N suppress tR1 in a boxA fusion, neither function can override more distal termination sites. (g) EJfect of hOS 1t f act ors on termination

at tR1

It has been suggested that boxA is a recognition site for nusA (Olson et al., 1982; Friedman & Olson, 1983) or nusB (D. Court, personal communication). If boxA and the Nus factors do indeed act at the same step(s) in the Nun termination reaction, we expect that inactivation of the Nus factors should mimic inactivation of boxA, and that the effects of inactivation of both together should be similar to those of inactivation of either one alone. In fact, we found that the nusA1, nusB5 and nusE71 mutations, like the boxA mutation, inverted the activity of Nun. Table 6 shows that Nun suppressed termination in the three nu,s mutant hosts. Although Nun enhancement of galactokinase synthesis was only about two-fold, it was reproducible and could represent nearly complete suppression of the inefficient tR1 terminator. Nun stimulation of galactokinase expression in the three nus mutants was not affected by the boxA mutation (Table 6). The phenotypic similarity of the nus and the boxA mutations is consistent with the idea that these reaction components act in concert, although other explanations are not ruled out,. In any event, it is clear that mutations in several of the known cis- and trans.acting elements in the N antitermination system convert Nun into a suppressor of tR1. We note that one nus mutation affects Nun differently from those just described. A temperaturesensitive lethal mutation in nusA, nusAtsl1 (Nakamura et al., 1986), reduces Nun termination in a boxA+ fusion, but does not transmute Nun into a suppressor of tR1 (data not’ shown). It may be that the NusAtsll protein is inactivated slowly after temperature shift. The nus mutat,ions also affect the growth of bacteriophage ;1 in the presence of Nun. 1 nin3 is no longer sensitive to Nun in nus mutant strains. Conversely, A boxA nin3 remains Nun dependent for growth (see section (f), above) in nus strains (data

641

not shown). These data indicate that Nun can form a complex with RNA polymerase even in the absence of wild-type Nus proteins. We do not know if these nus mutations allow Nun to establish a stable antitermination complex, but the properties of the boxA and boxA fusions suggest that this is not the case.

4. Discussion In this article we describe the isolation and characterization of mutations in the nutR locus of coliphage 2. The mutations were selected in a HK022 multilysogen carrying the chromosomal operon fusion: I pR-cro-nutR-tRl-gal. Expression of gal in this fusion is blocked by the HK022 Nun protein, which terminates transcription in the nutRtR1 region. Two mutants that expressed gal in the presence of active Nun were shown to carry transversions in the boxA (CGCTCTTA) sequence of nutR. One transversion, boxA (CTCTCTTA), was previously constructed in vitro (Olson et al., 1984) and shown to inhibit antitermination by 1 N protein. The other, boxAZ6 (CGCTATTA), is first described here. The mutations have similar phenotypes. (1) Both increase the efficiency of termination at or near tR1. The tR1 terminator is inefficient, terminating only about 50% of the pR-initiated transcripts. However, tR1 activity is strongly context’ dependent (Zuber et al., 1987). The efficiency of tR1 can reach 90% if the length of untranslated RNA upstream from the terminator is sufficiently large. We suggest that tR1 may be partially suppressed by the upstream boxA+ site, even in the absence of 2 N protein. An extended boxA-like sequence, derived from t,he rrnG operon, has recently been shown to suppress transcription termination (C. Squires CL. C. Squires, personal communication). In this model. the boxA and boxAl mutations inactivate boxA. resulting in increased tR1 activity. An alternative. that wild-type boxA does not suppress tR1, but that the boxA and boxA mutations stimulate tR1, is not ruled out (Zuber et al., 1987). Note that the relativelv short RNA region between cro and tR1 reacts with Rho and NusA, both of which affect termination at tR1 (Chen & Richardson, 1987; Faus et al., 1988) and boxA mutations might affect one or both of t.hese interactions. (2) Both boxA mutations invert the activity of Nun. Instead of provoking termination in the 2 nutR-tR1 region, Nun is required for gal expression in the mutant fusions. The boxA and boxA fusions are also activated by J. N-protein. although with reduced efficiency relative to boxA+. Analysis of in vivo RNA using Northern gels confirms the increased termination at tR1 caused by t’he boxA and boxA mutations, as well as the suppression of t,ermination by Nun. Our gels do not indicate the precise 3’ ends of the pR transcripts synthesized in the presence or absence of Nun. In fact, they appear to be of the same size. JXither Nun stimulates termination in the tR1 region, or tR1

642

K. Robledo et al.

terminated transcripts are processed in a 3’ to .i’ direction into the n&R region. These possibilities are under active investigation. When n&R carries the boxA mutation, neither Nun nor N can form a persistent antitermination complex. This was shown first by introducing gal Eoc polar mutations into the fusions. As previously demonstrated, N effectively suppresses the polarity of mutations promoter-distal to nutR+ (Adhya rt al.. 1974; Franklin, 1974). However, when the gaZ Eoc mutations are preceded by nutR boxA5, they remain polar in the presence of Nun or N. The failure of N to suppress terminators introduced downstream from nutR boxA confirms the results of other laboratories (Olson et al., 1984; D. Court, personal communication). Nun likewise fails to suppress polarity in the boxAlgaEEoc fusions. However, N clearly retains at least partial antitermination activity. This suggests that the nut sequences required for Nun termination and N antitermination activities may not fully overlap. The properties of the mutant fusions were reproduced in phage 1. We constructed the boxA and boxA variants of 2. N- nin3 and demonstrated that they grew only in the presence of Nun or N. Presumably, increased transcription termination at tR1 causes inadequate synthesis of 1 0 and P proteins; suppression of tR1 by Nun or N permits phage growth. A nin+ derivative of the boxA phage formed minute plaques, and required N for growth. Recause of the boxA mutation, the n®ion terminators are poorly suppressed by N. Nun is transmuted from a termination function to a suppressor of termination not only when boxA is mutated, but also if one of several host Nus proteins is altered. Thus, in contrast, to its activity in a wildtype cell, Nun stimulates gaZK expression from a n&R+ fusion in a nusA1, nusB5 or nusE7I mutant, strain. The common thread in these findings is that’ mutations that, inhibit antitermination by N, i.e. the boxA and the nus mutations, all invert Nun activity in the nutR-tR1 region. We suggest that under wild-type conditions, Nun, like N, interacts with nascent nutR RNA, RNA polymerase and Nus factors to form a complex. The boxA and nus mutations do not prevent the interaction of Nun with RNA polymerase, but change the nature of the complex formed. The mutant complex is capable of overriding tR1, but not distal terminators; either because of some unique feature of tR1. or because tR1 is very near to nutR, and the mutant complex is unstable. In other words, Nun interacts with RNA polymerase in the presence of wild-type factors to form a termination complex, and the RNA polymerase in the absence of wild-type factors to form a transient antitermination complex. In this respect, Nun and N are strikingly similar; the boxA and the nus mutations do not prevent an association between the phage proteins and RNA polymerase, but a persistent antitermination complex is not formed. Unlike its action at nutR, the nusA1 mutation

was shown to reduce. but not’ to eliminat~e, Sun termination at nutL (Robert et al., 1987). The basis of the difference between the nutL and nutR regions is not known. t,hese data, indicate that. a c-omplex Finally, between Nun and RNA polymerase can be formed at nutR even in the absence of wild-type Nus proteins, and/or wild-type boxA sequence. Our findings stress the similarity between termination and antitermination; a change in a single reaction component can change one reaction int*o the other. We art: grateful to P. DiMauro and I). Mills for the help provided in sequencing the hozA mutants, and T. Patterson and D. Court for sequencing the recombinant phage carrying the boxA mutation. We thank J. Oberto for sending us plasmid 50210. D. Friedman and D. Court for the strains provided, and Cathy and Craig Squires for many helpful discussions. This work was supported in part by NIH grant GM37219-03. References

Achord, 0. & Kennell,

D. (1974). ./. Mol. Hiol. 90. 581-599. Adhya, S. & Gottesman. MM. E. (1978). Annu. Rev. Biochem. 47. 967-996. Adhya, S.. Gottesman, M. E. & de Crombrugghe, K. (1974). Proc. Nat. Acad. Sci., C:.S.,4. 71. 2534-2538. Adhya. S., Gottesman. M. IX. & Court, D. (1977). .I. Mol. Biol. 112, 657-660. Aksoy. S. Squires, C. & Squires, C. (1984). -1. Hacteriol. 159. 260-264. Barik, S., Ghosh, B., Whalen. W.. Lazinski, 1). & Das. .4. (1987). Cell, 50, 885-899. Brosius, J. (1984). Gene, 27, 151-160. Chen, C.-Y. A. & Richardson. .J. P. (1987). .J. Hiol. Chem. 262. 11292-11299. Dambly-Chaudiere, C., Gottesman, M., Debouck, C. & Adhya, S. (1983). J. Mol. ilppl. Genet. 2. 46 56. Faus. I., Chen. C.-Y. A. 8: Richardson, ,J. 1’. (1988). J. Biol. Chem. 263, 10830~10835. Franklin. N. C. (1974). J. Mol. Biol. 89. 33-48. Friedman, D. I. 6 Gottesman, M. E. (1983). Lytic mode of lambda development. In Lambda II (Hendrix, R., Roberts J., Stahl F. & Weisberg, R., eds), pp. 21-51. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY Friedman, D. I. & Olson, E. (1983). Cell, 34. 143-149. Horwitz, R. ,J.. Li, J. & Greenblatt, *J. (1987). Cell. 51. 63 l-641 Leason, K. R. & Friedman, D. I. (1988). J. Bacterial. 170, 5051-5058. Malay, S. & Nunn, W. (1981). .I. Rncteriol. 145. 1110-1112. Maxam. 8. & Gilbert, W. (1980). Methods Enzymol. 65. 49%560.

Morgan, E. A. (1986). J. Bacterial. 168, l-5. Nakamura, Y., Misuzawa, S., Court, D. & Tsugawa, A. (1986). J. Mol. Biol. 189, 103-l 11. Olson, E., Flamm, E. & Friedman. D. I. (1982). (‘ell. 31, 61-70. Olson, E., Tomich, C. & Friedman. D. I. (1984). J. Mol. Biol. 180, 1053-1063. Robert, J., Sloan, S. B., Weisberg, R. A., Gottesman, M. E., Robledo, R. & Harbrecht, D. (1987). Cell, 51. 483492.

HK022 Nun Acts at Mutant 1 nutR Roberts, J. W. (1988). Cell, 52, 5-6. Salstrom, J. & Szybalski, W. (1978). J. Mol. Riot. 124, 195221. Sanger, F. Nicklen, S. & Coulson, A. R. (1977). Proc. Nut. Acad. Sci., U.S. A. 74, 5463-5467. Schauer, A. T., Carver, D. L., Bigelow, B., Baron, L. S. & Friedman, D. I. (1987). J. Mol. Biol. 194, 679-690.

643

Sharrock, R., Course, R. & Nomura, M. (1985). Proc. Nut. Acad. Sci., U.S.A. 82, 5275-5279. Silhavy, T.. Berman, M. & Enquist, L. (1984). Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Zuber, M., Patterson, T. A. & Court, D. L. (1987). Proc. Kat. Ad. Sci., lJ.S.A. 84, 45144518.

Edited by P. von Hippel