J. Mol.
Riol.
(1991) 220, 613-619
Escherichia coli Mutations That Block Transcription Termination by Phage HK022 Nun Protein R. Robledot,
B. L. Atkinson and M. E. Gottesman
Institute of Cancer Research Columbia University College of Physicians and Surgeons 701 West 168th Street, New York, NY 10032, U.S.A. (Received 16 October 1990; accepted 11 April
1991)
The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage J nut sites. Termination by Nun and antitermination by II N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at ;1 nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase p’ subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by 1 N function. The mutations antagonized Nun only at 1 nutR; termination at i nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the /?’ subunit of RNP may be Nun-specific. Keyujords: Nun termination;
phage HK022; transcription;
1. Introduction
boxA5, the nutR boxA mutation is specific to Nun termination (Robledo et al., 1990). This observation provided us with the incentive to select host mutations that blocked Nun termination, and then to determine their effects on N antitermination. We report below the isolation of mutations in nusA, nusB, nusE and rpoC that block Nun termination at 1 nutR. The nusA mutation and the three rpoC mutations are Nun-specific; N antitermination is functional. Furthermore, the mutations affect Nun action at nutR, and do not block termination at nutL.
The Nun product of phage HK022 and the N product of phage ,I act antagonistically on the A chromosome (Robert et al., 1987). Nun provokes termination at or near the A nut sites, whereas N modifies RNA polymerase at these sequences to suppress termination at distal loci (see Friedman & Gottesman, 1983). Although their final results are diametrically opposed, the two reactions share common features, Nun and N are to some extent homologous, and are located in analogous regions of their respective phage chromosomes (Oberto et al., 1989; Lazinski et al., 1989). Mutations in the Iz nut sequences that block N antitermination can also prevent Nun termination. Mutations at n&L that suppress both Nun and N lie in the boxB region; at nutR, the boxA mutation affects both functions (Olson et al., 1984; Robert et al., 1987; Robledo et al., 1990). Similarly, host mutations isolated as preventing antitermination by N also reduce termination by Nun. These mutations, nusA1, nusB5 and nusE?‘Z (see Friedman et al., 1984), suppress to various degrees Nun termination at nutL and nutR (Robert et al., 1987; Robledo et al., 1990). The requirements of the two reactions do not, however, completely overlap. Thus, in contrast to t Present address: Istituto Universita di Sassari. Viale Sassari. Italy.
di Microbiologia, San Pietro 43/B,
Nus factors; RNA polymrrase
2. Materials and Methods (a) Bacteriological
techniques
Standard media and bacteriological techniques were employed (Silhavy et al., 1984; Robledo et al., 1990). Cloning and other DNA manipulations were as described (Sambrook et al., 1989). (b) Bacteria
and
phege
N99 is a W3102 galK2 strR strain. Strain 6D7 carries a ApR-cro-nutR-tRl-gal operon fusion (Dambly-Chaudiere et aE., 1983). The pR fusion strain, R35, was constructed as follows: A lzad :: TnlO mutation was introduced in strain 6D7 by phage Pl transduction. The fusion was then transferred to N99 by Pl transduction, selecting for TetR Gal+ transductants. Finally, the TnZO was eliminated by selecting for Nad+ on minimal medium. Strain pu’6208 carries a IpL-nutL-la& operon fusion (Robledo et al.,
07100 613
0022%2836/91/150613-07
$03.00/0
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Press Limited
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614
1990). Strains carrying TnlO elements linked to nusA, nusB and nusE were provided by D. Friedman, who also supplied strain K3959, which carries the nusA gene of Salmonella typhimurium. Phage HK022, limm434, Limm434nin5 and limm434r32 are from the NIH or Columbia collections. (c) Plasmids Plasmid PJO210 is a pBR322 derivative expressing HK022 Nun (Oberto et al., 1989); plasmid pNAS200, a low copy number plasmid based on the incFI1 plasmid NRl, expresses I N from a plac promoter (Schauer et al., 1987). Plasmid pMK16 is TetR, KanR and is incompatible with pBR322 (a gift from D. Mills). Plasmids expressing Escherichia coli nusB or nusE genes were provided by D. Friedman, who also provided the following pBR322-based plasmids: pNAG15 (Am R TetS expressing E. COBnusA); pNAS1322 (AmpR Tet P expressing the nusA gene of S. typhimurium); pNAX1 (AmpR TetR expressing nusA hyl); pNAX2 (AmpR TetR expressing nusA hy2). The 5’ two-thirds of the nusA hyl gene is derived from E. coli and the remainder from S. typhimurium; the nusA hy2 gene is the reciprocal recombinant. Plasmid expressing rpoB and/or rpoC were obtained from A. Goldfarb and J. Lee. These include pGB218 expressing rpoB and rpoC (Barry et al., 1979), pXT7fl expressing rpoB (McKinney et al., 1987) and pT7fl expressing rpd: (Zalenskaya et al., 1990). The rpoC gene is cloned as an SphI-SspI fragment downstream from the phage T7 promoter. The fragment carries only 94 bpt of DNA upstream from rpoC including the weak P4 promoter (from which the gene is expressed) and a small portion of rpoB. The fragment terminates 144 bp distal to rpoC. (d) Enzyme assays Galactokinase and fl-galactosidase assays were performed as described (Robledo et al., 1990). (e) Amplijkation
of chromosomal DNA by PCR
A 1.6 kb E. coli chromosomal DNA fragment including nusAlO0 was amplified by the polymerase chain reaction (PCR) (Saiki et al., 1988). The amplification reaction was carried out for 30 cycles in the DNA Thermal Cycler (Perkin Elmer-Cetus) in a 100 ~1 final volume under the following conditions: the DNA was denatured at 94°C for 1 min, annealed at 60°C for 2 min with oligonucleotide primers (co-ordinates 795 to 819, 5’ to 3’, top strand; coordinates 2393 to 2417, 3’ to 5, bottom strand) and extended at 72°C for 3 min. To analyze the amplification product, a sample of the reaction mixture was subjected to electrophoresis on a 1% agarose gel and stained with ethidium bromide. The amplified DNA was purified by extraction with phenol and precipitation with ethanol, and analyzed by digestion with restriction endonuclease prior to cloning and sequencing. (f) Cloning the nusAlO0
mutation
The oligonucleotides used for amplification represented the nusA sequences described in section (e), above, and carried a 3’ tail bearing an EcoRI sequence. After purification, the amplified material was digested with EcoRI and t Abbreviations used: bp, base-pairs; PCR, polymerase chain reaction; kb, 10’ bases or base-pairs.
ligated into a lZapI1 vector predigested with EcoRI and dephosphorylated (from Stratagene Inc., La Jolla, CA). After packaging, white recombinant plaques were detected on IPTG/X-gal (isopropyl-/&n-thiogalactoside/ 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside) plates using strain XLl-Blue. Plasmid DNA was excised from the 1 clones in vivo, and isolated for sequencing (protocol from Stratagene Inc.). (g) DNA sequencing Double-stranded plasmid DNA was sequenced by the dideoxy method (Sanger et al., 1977), using Sequenase version 2.0 and following the protocol provided by the United States Biochemical Co. DNA was labeled with [“S]dATP at 1000 Ci/mmol (Amersham Inc.). Sequencing reactions were fractionated on 6% polyacrylgels, and run at a constant power of amide/7 M-Urea 70 W. After electrophoresis, the gels were fixed for 20 min with 10% (v/v) acetic acid, 12% (v/v) methanol. dried under vacuum for 1 h and autoradiographed at room temperature using Kodak XAR5 films.
3. Results (a) Isolation
and screening of Nun-resistant E. coli mutants
The nun gene product of HK022 blocks the expression of genes in the phage 1 pL and pR operons by terminating transcription at or near the n&L and n&R sites, respectively (see Fig. 1). E. coli strain R35 is an N99 derivative bearing a pR operon fusion to gal (from strain 6D7; Dambly-Chaudiere et al., 1983). The fusion has the structure: lcI857oRpR-cro-n&R-tRl-galETK R35 is Gall at 32”C,
(Fig. 1). since pR
is negatively
controlled by the ~1857 repressor. At 42”C, the temperature-sensitive repressor is denatured; the fusion is activated, and the strain becomes Gal+. The expression of gal is N-independent, since the efficiency of tR1 is approximately 50% (Robledo et al., 1990). In the presence of Nun, R35 is Gal- at all temperatures. By selecting from Gal+ colonies, we obtained mutants resistant to Nun termination. Mutations in nutR have been described (Robledo et al., 1990). Here we describe trans-acting mutations that identify
host factors
loci-t-N-nun-pL
required
~1657
for Nun activity.
pR-cro-nutR-tt?l-qoETK
(al n&R
CGCTCTTA cAcAl-T’ccA GCCC~AAGGGC
IWfL
CGCTClTA aAoATTa A GCCCTWAGGGC (b)
Figure 1. The 1 nutL and nutR fusions. (a) The 1 pL and I pR operons are shown linked to reporter genes. The direction of transcription is indicated by the broken arrows. The pL fusion carries uncharacterized weak terminators. The pR fusion includes the tR1 terminator, which, in its normal context, is -50% efficient. In strain N6028, which carries the pR fusion, the N gene is inactivated by amber mutations. The strains were described in detail by Robert et al. (1987). (b) The 1 nutR and I nutL Common nucleotides are sequences are compared. capitalized.
Nun Termination Spontaneous Gal+ derivatives of R35 carrying the Nun plasmid pJO210 (Oberto et al., 1989) were isolated on MacConkey galactose indicator plates at 42°C and screened for their ability to support the growth of ;limm434nin5. This hybrid phage carries the nut sites of L and is sensitive to Nun. The nin5 deletion removes the tR2 and tR3 terminators (Leason & Friedman, 1988), and allows the phage to grow in the absence of N, a pL operon function. In principle, therefore, a host mutation that blocked Nun termination at n&R would permit growth of limm434nin5. The screen yielded six independent mutants. To demonstrate the presence of active Nun in these mutants, we cured plasmid pJO210 by selecting transformants of the incompatible plasmid pMKl6, and then reintroduced pJO210. The resultant transformants remained Gal+, eliminating the possibility of a mutation in nun. A genetic analysis of the strains was then begun, to locate the mutant host genes. (b) Mapping
the host mutations
Since the nusA1, nusB5 and nusEY1 mutabions reduce Nun termination (Robert et al., 1987; Robledo et al., 1990), we first asked if our mutations were locat,ed in a nus gene. Using linked TnlO markers, we introduced the wild-type alleles of nusA, nusB, nusC (rpoB), nusD (rho) and nusE into the mutants by Pl transduction. The tetracyclineresistant transductants were then screened for their Gal phenotype and for sensitivity to superinfecting Aimm434nin5. Replacement of a mutant nus with a nus+ allele will yield a Gal- transductant resistant to kmm434nin5. By this means, we located one mutation each in nusA, nusB and nusE. Three mutations were mapped to the nusC region; they are, however, located in rpoC rather than rpoB (see below). The mutations were than moved into strain R35 lysogenic for HK022 by Pl cotransduction with the
Table 1 C’omplementation of host mutants Plasmid Host Wild-type nusAlO0 nusBlO0 nusEIO0 rpoCIO0 rpOCl01 rpoc102
None
nus+
o-3( +02) 7.3(+01) &0(+02) 47( f02) 64( f 1.0) 7.0( + 0.3) 7.7( 50.1)
@4( kO.3) @l( kO.1) @8( + @2) l.O(kO.1) l.l( kO.1) 1.3( kO.1)
in Phage HKOZZ
615
linked TnZO marker. Gal+ transductants were isolated for all six mutations, suggesting that each mutation represented a single genetic locus. The transductants were then transformed with plasmids expressing wild-type nus genes and the expression of galactokinase was determined. In the case of the putative nusA, nu.sB and nusE mutants, the wildtype gene, expressed from a multicopy plasmid, restored Nun activity (Table 1). As a control, mutants were also transformed with the other nus plasmids. The nusA mutant transformed with plasmids expressing nusB or nusE retained a mutant phenotype. Similarly, we saw no complementation when the nusB mutant was transformed with the nusA or nusE plasmids, or when the nusE mutant was transformed with nusA or nusB plasmids (data not shown). These mutants will be referred to as nusA100, nusBlO0 and nusE100. The nusC region mutations were suppressed by a plasmid (pT7j’; see Materials and Methods) that expresses rpoC (Table 1). The E. coZi DNA fragment cloned in pT7/I’ carries only 94 bp of DNA upstream and 144 bp downstream from rpoC; it is, therefore, unlikely to encode a gene in addition to rpoC. Plasmid pT7j did not suppress the nusAZ00 mutation, indicating that rpoC overexpression is not a general suppressor of nus mutations (data not shown). As shown by their Gal phenotypes, the nusC region mutations were also suppressed by pGB218, which expresses rpoB and rpoC, but not with pXT7/?, which expresses only rpoB (data not shown). These results indicate that the nusC region mutations lie in rpoC, rather than rpoB. We shall refer to these mutations as rpoClO0, rpoClO2 and rpoC102. To support further the map assignment of the we performed marker rescue rpoC mutations, experiments. The rpoC mutant strains bearing plasmid pT7/I’ are white (Gal-) on McConkey galactose indicator plates; rpoC+ expressed in multi-copy is dominant! to the chromosomal rpoC mutations. Incubation of these strains for two days at 42°C yielded red (Gal+) papillae at lOO-fold higher levels than the R35 HK022 lysogen. We presume that the Gal’ derivatives represent transfer of the rpoC mutations from the chromosome to the plasmid. This is the first evidence linking the /?’ subunit of RNP to Nun termination. (c) Effect of host mutations on I growth
Fusion: pR-cro-nutR-tRl-qalETK Values represent average galactokinase units from duplicate samples determined 1 h after shift from 32°C to 42°C (see Materials and Methods). Fluctuations are given in parentheses. The wild-type strain is R35 lysogenie for HK022; mutant alleles were introduced into the wild-type strain by PI transduction. The complementing nusA+, nusB+, nuaE+ and rpoC+ plasmids are described in Materials and Methods.
Classic E. cob nus mutations block N antitermination. As a consequence, 1 fails to propagate on nus mutants. To determine if our nus and rpoC mutations inhibited N antitermination, we plated &mm434 on the mutant strains at 42°C. The nusB100 mutation totally blocked phage growth. The nusElO0 mutation reduced the efficiency of limm434 plating. In contrast, the nusA100, rpoClO0, rpoClO1 and rpoClO2 mutants supported phage growth as well as wild-type strains (data not shown). Recall that all mutant strains were permissive for the N-independent &mm434 variant,
R. Robledo et al.
616
Table 2
Table 3
Activities of HK02.2 Nun and Lambda N at nutR
Activities of HK022 Nun and Lambda N at nutL
Plasmid Host Wild-type nusAIO0 nusBlO0 rpoclO0 rpoClO1 rpoC1OP
Plasmid
None
PN
pNun
45( f 0.9) 31( k9.1) 51(&@2) 3.9( 5 04) 44( 59.1) 57(+@6)
138( * 1.0) 1@5( + 94) 57( f05) 11.9( k 1.6) 123( +@8) 137( ko.2)
@7( kO.4) 7.2( f 0.6) 7.8( k @9) 6%( kO.3) &9( f 94) %9( * 0.7)
Fusion: pR-cro-nutR-tRl-galETK Galactokinase units were determined as for Table 1. Plasmid pN is pNAS299; plasmid pNun is pJO210. All strains are isogenic with R35 (wild-type).
limm434nin5. These results show that host mutations that block Nun termination do not necessarily also inhibit N antitermination. (d) Nun suppresses termination host mutants
at tR1 in
The nusA1, nusB5 and nusE71 mutations do not only abolish the termination activity of Nun, but invert Nun to a suppressor of the i tR1 terminator (Robledo et al., 1990). We show in Table 2 that the nusAl00 and rpoC mutations also reduce termination at tR1. In this experiment, we measured the galactokinase activity after induction of a pR-nutRtRl-galK chromosomal fusion. Termination at tR1 was as efficient as wild-type, approximately 50 o/o1in all mutant strains tested. Consistent with their phenotype for Iz growth, N antitermination at tR1 was unaffected by the nusA100 and rpoC mutations, but was blocked by the nusBlO0 mutation. The nusAlO0 mutation, the rpoC mutations, and possibly the nusBlO0 mutation, all inverted Nun to a weak suppressor of termination. (e) The host mutations are nut-speci$c Nun terminates transcription at or near the A nutR and the 1 nutL sequences (Robert et al., 1987). The two nut sequences are not perfectly homologous, and they differ in their locations relative to their cognate promoters and terminators. To determine if the host mutations also blocked Nun termination at nutL, we assayed fi-galactosidase expressed from a chromosomal pL-nutL-1acZ fusion (Table 3). In contrast to their effects at nutR, the mutations did not prevent Nun termination at nutL (Table 3, column 3), although the rpoClO1, rpoClO2 and possibly several of the other host mutations did reduce its efficiency. In the case of rpoClO2, the reduction in Nun termination has physiological consequences for the growth of A (see below). We found that the pL-nutL-ZacZ fusion was less active in all host mutations tested (Table 3, column 1). Perhaps the mutations increased the efficiency of weak termination sites within the fusion. N strongly
Host
None
PN
pNun
Wild-type nu.sAlOO nusBlO0 rp0C100 rpoClO1 rpoc1oe
1181 693 648 .554 574 441
4697 2837 653 3184 3483 3575
51 86 122 53 92 117
Fusion: pL-nutL-lad Values represent average fi-galactosidase units from duplicate samples determined 1 h after shift from 32°C to 42°C (see Materials and Methods). Fluctuation was 22% or less. All strains are isogenic with N6208 (wild-type).
stimulated the expression of ZacZ in wild-type hosts, and, with the exception of nusBlO0, in the mutant cells as well (Table 3, column 2; nusEZO0 was not assayed). (f) Why i is Nun-resistant in nusAlO0 and rpoC mutants The termination activity of Nun at nutL in the nusAlO0 and rpoC strains was surprising. The N gene lies in the pL operon just promoter-distal to n&L, and will not be expressed when Nun is active. N is essential for the growth of wild-type J. ‘and limm434. Why, then, is I Nun-resistant in these strains (see above)? The answer to this apparent paradox came from the analysis of the J. variant, Ar32. The r32 mutation consists of an IS2 insertion between tR2 and the 1 cII gene (Daniels et al., 1983). IS2 includes a strong rho-dependent terminator, which in the absence of N antitermination, completely prevents transcription of the downstream I 0 and P replication genes. Unlike wild-type A, IX?2 cannot replicate without N. Table 4 shows that 1r32 did not propagate on nusA100 and the rpoClO0 mutants in the presence of Nun (Table 4, column 2).
Table 4 Effect of host mutations on growth of I and lr32 Host Wild-type Wild-type/pNun nusAlO0 nwAlOO/pNun rpoC100 rpoClOO/pNun rpoClO2 rpoC102/pNun
I
h32
++ ++ ++ ++ ++ ++ ++
++ ++ ++ f-lf
1, -N-n&L-pL-cl-pR-cro-n&R-tRI-cII-0-P-tRP-tR3-Q ~~ PnutR-tRl-IS2-cII-O-P---1732, Phage are 2imm434. They were plated on wild-type (N99) or on isogenic mutant strains at 42°C. + + . normal growth; +, minute plaques; - , no growth.
Nun Termination The lr32 variant was partially resistant to Nun in rpoC102 mutants. Recall that the rpoC102 mutation reduced Nun termination efficiency in the pL operon (Table 3). We explain this result as follows. The nusA100 and rpoC mutations permit, wild-type 1 to express ,J genes 0 and P and to replicate in the presence of Nun. The replication of 1 results in the titration of Nun. N is then expressed, and N acts to antiterminate the distal tR2 and tR3 terminators in the nin region, allowing plaque formation. Since the Ar32 variant cannot replicate without N, Nun is not titrated, and the pL operon, including N, cannot be expressed. The growth of ir32 is blocked by termination at IS2. (R) The nusASa’ gene blocks Nun termination and N antitermination The existence of a nusA allele that specifically blocked Nun termination prompted us to examine the effe& of the Salmonella nusA gene on Nun termination in the pR and pL operons. NusASa’ does not support N antitermination (Friedman & Baron, 1974). We introduced nusAS”’ into E. coli by PI transduction and measured the expression of pRnutR-galK and pL-n&L-1acZ fusions. We found that the nusAS”’ allele blocked Nun inhibition and N stimulation of both the pR and pL fusions (Table 5). Tn addition, Nun stimulated the pR fusion suggesting that nusASa’ may also invert Nun into a weak suppressor of tR1. (h) Fine
m.apping of the nusAlO0 mutation
To map the nusA100 mutation, we first located the portion of the nusA gene required for Nun termination. Strains carrying hybrid E. colil Sal~monelln nusA genes have been constructed and tested for A plaque formation. A hybrid gene in which the 5’ two-thirds is derived from E. coli (hy-1) supports E, growth, whereas the reciprocal hybrid (hy-2) does not (A. Cranston, unpublished results,
Table 5 NusASa’ blocks both N antitermination Nun termination
and
617
in Phage HK022
1988). Two E. coli nusA cited by Friedman, mutations, nusA1 and nusAtsl1, lie in the 5’ twothirds of nusA: inhibition of both Nun termination and N antitermination by nusA 1 has been described (Robert et al., 1987; Robledo et al., 1990). To confirm the role of the amino-terminal portion of nusA in Nun termination, we introduced plasmids carrying nusAE, “‘li, nusAS”‘, nus4 hy-1 or nusA hy-2 into HK022 lysogens, and measured the activity of the pR-nutR-galK fusion. As expected, Nun blocked galK expression in strains bearing nusAE. “li or nusA hy-I, but not in nusASa’ or nusA hy-2 strains (data not shown). Assuming that the plasmid-born nusA alleles are dominant to the chromosomal nusA+, these results indicate that the 5’ two-thirds of the E. coli nusA protein is required for both N antitermination and Nun termination. (i) The sequence of nusA 100 As the first step in sequencing nusA100, the chromosomal allele was amplified by PCR and transferred to plasmid pBR322 (see Materials and Methods). Distance tests revealed that whichever nusA allele was present on pBR322, it was dominant for Nun termination (data not shown). This allowed us to locate the nusA100 mutation in a 778 bp AatII-NcoI fragment derived from the amino-terminal two-thirds of nusA. Replacement of the wild-type AatII-NcoI fragment with a fragment cloned from nusA100 yielded pBR322 clones conferring the mutant phenotype (data not shown). Conversely, substitution of the mutant fragment with wild-type converted a nusAlO0 plasmid clone into one with a wild-type phenotype. The sequence of nusA100 is shown in Table 6. We find a single base substitution, a G to A transition. The mutation converts codon 136 from glutamic acid to lysine. We note that the nusAlO0 mutation is located relatively far from the nusA1 (codon 183) and nusAtsl1 (codon 181) mutations and the arginine-rich domain (amino acids 170-I 91). In addition, we find four changes from the published nusA sequence (Ishii et al., 1984; Saito et al., 1986). These changes were detected independently by two groups (Craven & Friedman, 1991,
Table 6
Plasmid
Map of nusA mutations
Fusion
?&US A
None
PN
pNun
pR-nutR-galK pR-nutR-galK pL-n&L-lad pL-nutLlaci5
E. coli Sal E. coli ‘Sd
2.4( &@I) 2.3( + 63) 535 300
8.3( +@2) 3.5( f 0.2) 2156 229
@l(+O.l) 52( * 0.2) 21 305
Wild-type:
The pR and pL fusions are present in strains 6D7 and N6208, respectively. The nusA gene of S. typhimurium was introduced into both strains by PI transduction (see Materials and Methods). Values given in the top two lines represent galactokinase units as determined for Table 1; fluctuations are shown in parentheses. Reta-galactosidase units, shown in lines 3 and 4, were determined as for Table 3. Fluctuations in /?-galactosidase values were l3o/, or less.
AatIl 44
GAA Glu 136 ___
GM’ Gly 181 ___
(‘T(’
NC01
LeU
183 -
304
Mutants: AAA Lys nusAlO0
(:A(’ Asp nusAl1
( ‘G( ( k n~sA1
Codon sequences and amino acids of nusA mutations (bottom) compared to wild-type (top). Amino acid positions are counted from the translation start site of nusA. Restriction sites used for cloning are indicated.
618
R. Rob&o et al.
and Ito et al., 1991) and are in agreement with our sequence.
4. Discussion In this paper we report the isolation and characterization of E. coli mutations that block the activity of the phage HK022 Nun function. Nun provokes termination at or near the I n&R and nutL sequences. Our selection strategy demanded expression of gal from a JpR-nutR-tRl-gal operon fusion controlled by the lcI857 repressor. E. coli carrying this fusion are Gal- when Nun is active. Our selection for Gal+ colonies at 42°C yielded mutations in nusA, nusB and nusE, genes previously shown to play a role in both N antitermination and Nun termination (see Friedman & Gottesman, 1983; Friedman et al., 1984; Barik et al., 1987; Robert et al., 1987). In addition, we isolated three independent mutations in rpoC, the structural gene for the jl’ subunit of RNP. Unlike the well-documented role of the fl subunit of RNP (Jin et al., 1988), the involvement of the fl subunit in transcription termination has only recently been proposed. Evidence for this involvement is based on the isolation of rpoC suppressors of the rho201 (Jin & Gross, 1989) and the nusAtsl1 mutations (Ito et al., 1991). In addition, the 3’ nucleotide of in vitro paused transcripts can be cross-linked specifically to the p’ subunit (A. Goldfarb, personal communication). Our data implicate the /Y subunit in the termination reaction promoted by Nun factor. The rpoC mutations block Nun termination but not antitermination by 1 N function. In contrast, the rpoB mutation, nusC60, blocks N antitermination but not Nun termination (see Friedman & Gottesman, 1983; R.R., B.L.A. & M.E.G., unpublished results). The nusAlO0 mutation also specifically blocks Nun termination without affecting N antitermination. The 1 variant lr32, whose requirement for N antitermination is more stringent than wild-type A, grew normally in the nusAlO0 (and rpoC) mutants. Recall that the nusA1 mutation inhibits both N and Nun reactions (Robledo et al., 1990). Thus the elements of NusA needed for Nun termination and N antitermination are overlapping rather than identical. The nusA100 mutation represents a G to A transition at position 1260 resulting in a glutamic acid to lysine change at codon 136 within a revised nusA sequence. Like the nusA1 and nusAtsl1 mutations, nusAlO0 resides in the 5’ two-thirds of nusA. The nusAlO0 mutation lies 140 bp promoter-proximal to nusA1. Apart from its effect on Nun termination at nutR, nusAlO0 confers no detectable phenotype on its host. Our E. coli mutations also inverted Nun into a suppressor of tRI, an effect we have previously noted for the nusA1, nusB5 and nusE71 mutations, and for point mutations in the boxA sequence of nutR. We have yet to isolate a mutation that
confers a Nun null phenotype. Perhaps such mutants are temperature-sensitive to growth, and would, therefore, be eliminated in our selection protocol. We have selected host mutations that suppressed the Nun termination at nutR. When tested for Nun termination at nutL, the mutations were ineffective. The two nut sites differ in sequence, as do tL1 and tR1. The close location of nutR to tR1 is not duplicated at nutL. Which of these differences is relevant to the Nun phenotype is unknown. A selection based on suppression of Nun at nutL has not yet yielded bacterial mutants. Recall that the nusB5 and nusE71 mutations allowed 50% readthrough of nutL in the presence of Nun. The nusA1 mutation was less effective in blocking Nun termination, but still permitted significant readthrough (Robert et al., 1987). These nus mutations were selected as preventing N termination in both the pL and pR operons. Whether host mutations selected for N inactivation at nutL or nutR might be nut specific is an open question. We propose that after transcription of the I nut region, RNP, in concert with NusA, NusB and NusE, can be engaged by the I N or HK022 Nun gene product. Engagement by N produces an antitermination complex, whereas Nun engagement normally leads to rapid termination. Mutations in n&R boxA or in the Nus factors can produce a Nun complex capable of suppressing transcription termination at tR1. Possibly, the mutant Nun complex is short lived, since suppression of distal terminators is not observed (Robledo et al., 1990). Finally, and perhaps most importantly, our mutational analysis suggests that interactions between N and the b subunit are involved in formation of the antitermination complex, whereas Nun interactions with /I play a role in the termination complex formation. We thank U. Beauchamp for oligonucleotide preparations and for her help in sequencing the nusA gene. We are also grateful to Dr D. Friedman for various strains and plasmids, to Jookyung Lee for rpoB and rpoC plasmids, and to Dr R. Weisberg and S. Sullivan for criticisms and helpful discussions. This work was supported in part by NIH grant GM37219-03.
References Barik, S., Ghosh, B., Whalen, W., Lazinski, D. & Das. A. (1987). An antitermination protein engages the elongation transcription apparatus at a promoter-proximal recognition site. CeEl, 50, 885-899. Barry, G., Squires, C. L. & Squires, C. (1979). Control features within the rplJL-rpoBC transcription unit of Escherichia coli. Proc. Nat. Acad. Sci., U.S.A. 76. 4922-4926. Craven, M. G. t Friedman, D. I. (1991). Analysis of the Escherichia coEi nusAl0 (Cs) allele: relating nucleotide changes to phenotypes. J. BacterioE. 173, 1485-1491. Dambly-Chaudiere, C., Gottesman, M. E. Debouck, C. & Adhya, S. (1983). Regulation. of the pR operon of bacteriophage lambda. J. Mol. Appl. Cenet. 2, 45-56. Daniels, D. L., Schroeder, J. L., Szybalski, W., Sanger. F., Coulson. A. R., Hong, G. F., Hill, D. F..
Nun Termination Petersen, G. B. & Blattner, F. R. (1983). Complete annotated lambda sequence. In Lambda ZZ (Hendrix. R., Roberts, J., Stahl, F. t Weisberg, R., eds). pp. 519-676, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, KY. Friedman, D. I. (1988). Regulation of phage gene expression by termination and antitermination of transcription. In The Bacteriophages, vol. 2. pp. 263-319, Plenum Publishing Corp.. Rjew York. Friedman. D. I. & Baron, L. S. (1974). Genetic characterization of a bacterial locus involved in the activity of the X function of phage lambda. Virology, 58. 141- 148. Friedman, 11. I. & Gottesman, M. E. (1983). Lytic mode of lambda development. In Lambda ZZ, pp. 21-51. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, SY. Friedman, 11.I.. Olson, E. R., Georgopoulos, C.. Tilly. K.. Herskowitz. I. & Banuett. F. (1984). Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbial Rev. 48. 299-325. Ishii. S.. Ihara. M.. Maekawa, T., Xakamura, Y., Uchida. H. & Imamoto, F. (1984). The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia roli. Nucl. Acids Res. 12, 3333-3342. Ito. K., Egawa~ K. & Xakamura, Y. (1991). Genetic interaction between the /?’ subunit of RNA polomerase and the arginine-rich domain of EscherichLa coli nusA protein. J. Bacterial. 173, 1492-1501. Jin, D. J. & Gross. (1. A. (1989). Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants. Mol. Gen. &net. 216, 269-275. Jin, I). ,J.. Walter, W. A. & Gross, C. A. (1988). Charact,erization of the termination phenotypes of refampicin-resistant mutants. J. Mol. Biol. 202, 245-253. Lazinski, I).. Grzadzielska, E. & Das, A. (1989). Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell, 59, 207-218. Leason. K. R. dz Friedman, D. 1. (1988). Analysis of transcription termination signals in the nin region of bacteriophage lambda: the rot deletion. J. Bacterial. 170. 505lm--5058. McKinney. .J. D.. Lee, J., O’Neill, R. E. 6 Goldfarb, A. (1987). Overexpression and purification of a biologicall3 active rifampicin-resistant /I subunit of Eacherichia coli RPU’A polymerase. Gene, 58, 13-18.
in Phuge HK022
619
Oberto, J., Weisberg, R. A. & Gottesman, M. E. (1989). Structure and function of the nun gene and the immunity region of the lambdoid phage HK022. J. Mol. Biol. 207, 675-693. Olson, E., Tomich, C. & Friedman, D. I. (1984). The nusA recognition site. Alteration in its sequence or position relative to upstream translation interferes with the action of the X antitermination function of phage lambda. J. Mol. Biol. 180, 1053-1063. Robert, J., Sloan, S. B., Weisberg, R. A., Gottesman, M. E., Robledo, R. & Harbrecht, 1). (1987). The remarkable specificity of a new transcription termination factor suggests that the mechanism of termination and antitermination are similar. Call, 51, 483-492. Robledo, R., Gottesman, M. E. & Weisberg, R. A. (1990). 1 nut R mutations convert HK022 Xun protein from a transcription termination factor to a suppressor of termination. J. Mol. Biol. 212, 635-643. Saiki, R. K., Gelfand, D. H., Stoffel, S.. Scharf, S. J.. Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988). Primer-directed enzymatic ampliiication of DNA with a thermostable DNA polymerase. Science, 239, 487-491. Saito, M., Tsugawa, A., Egawa, K. & Xakamura, Y. (1986). Revised sequence of the nusA gene of Escherichia coli and identification of nusA I1 (ts) and nusA1 mutations which cause changes in a hydrophobic amino acid cluster. Mol. C&n. Genet. 205. 380-382. Sambrook, J. F., Fritsch, E. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Sanger. F.. Nicklen, S. & Coulson, A. R. (1977). DSA sequencing with chain-terminating inhibitors. Proc. Nat. A cad. Sci., U.S. A. 74, 5463-5467. Schauer, A. T., Carver, D. L., Bigelow, B., Baron, L. 6. & Friedman, D. I. (1987). 11v antitermination system: functional analysis of phage interactions with the host NusA protein. J. Mol. Biol. 194, 679-690. Silhavy, T. J., Berman, M. L. & Enquist. L. W. (1984). Experiments in Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Zalenskaya, K., Lee, J., Gujuluva, C. X.. Shin, Y. K., Slutsky, M. & Goldfarb, A. (1990). Recombinant RNA polymerase: inducible overexpression, purification and assembly of Esch,erichia coli rpo gene products. Gene, 89, 7-12.
Edited by P. von Hippel
Riol.
(1991) 220, 613-619
Escherichia coli Mutations That Block Transcription Termination by Phage HK022 Nun Protein R. Robledot,
B. L. Atkinson and M. E. Gottesman
Institute of Cancer Research Columbia University College of Physicians and Surgeons 701 West 168th Street, New York, NY 10032, U.S.A. (Received 16 October 1990; accepted 11 April
1991)
The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage J nut sites. Termination by Nun and antitermination by II N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at ;1 nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase p’ subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by 1 N function. The mutations antagonized Nun only at 1 nutR; termination at i nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the /?’ subunit of RNP may be Nun-specific. Keyujords: Nun termination;
phage HK022; transcription;
1. Introduction
boxA5, the nutR boxA mutation is specific to Nun termination (Robledo et al., 1990). This observation provided us with the incentive to select host mutations that blocked Nun termination, and then to determine their effects on N antitermination. We report below the isolation of mutations in nusA, nusB, nusE and rpoC that block Nun termination at 1 nutR. The nusA mutation and the three rpoC mutations are Nun-specific; N antitermination is functional. Furthermore, the mutations affect Nun action at nutR, and do not block termination at nutL.
The Nun product of phage HK022 and the N product of phage ,I act antagonistically on the A chromosome (Robert et al., 1987). Nun provokes termination at or near the A nut sites, whereas N modifies RNA polymerase at these sequences to suppress termination at distal loci (see Friedman & Gottesman, 1983). Although their final results are diametrically opposed, the two reactions share common features, Nun and N are to some extent homologous, and are located in analogous regions of their respective phage chromosomes (Oberto et al., 1989; Lazinski et al., 1989). Mutations in the Iz nut sequences that block N antitermination can also prevent Nun termination. Mutations at n&L that suppress both Nun and N lie in the boxB region; at nutR, the boxA mutation affects both functions (Olson et al., 1984; Robert et al., 1987; Robledo et al., 1990). Similarly, host mutations isolated as preventing antitermination by N also reduce termination by Nun. These mutations, nusA1, nusB5 and nusE?‘Z (see Friedman et al., 1984), suppress to various degrees Nun termination at nutL and nutR (Robert et al., 1987; Robledo et al., 1990). The requirements of the two reactions do not, however, completely overlap. Thus, in contrast to t Present address: Istituto Universita di Sassari. Viale Sassari. Italy.
di Microbiologia, San Pietro 43/B,
Nus factors; RNA polymrrase
2. Materials and Methods (a) Bacteriological
techniques
Standard media and bacteriological techniques were employed (Silhavy et al., 1984; Robledo et al., 1990). Cloning and other DNA manipulations were as described (Sambrook et al., 1989). (b) Bacteria
and
phege
N99 is a W3102 galK2 strR strain. Strain 6D7 carries a ApR-cro-nutR-tRl-gal operon fusion (Dambly-Chaudiere et aE., 1983). The pR fusion strain, R35, was constructed as follows: A lzad :: TnlO mutation was introduced in strain 6D7 by phage Pl transduction. The fusion was then transferred to N99 by Pl transduction, selecting for TetR Gal+ transductants. Finally, the TnZO was eliminated by selecting for Nad+ on minimal medium. Strain pu’6208 carries a IpL-nutL-la& operon fusion (Robledo et al.,
07100 613
0022%2836/91/150613-07
$03.00/0
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Press Limited
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614
1990). Strains carrying TnlO elements linked to nusA, nusB and nusE were provided by D. Friedman, who also supplied strain K3959, which carries the nusA gene of Salmonella typhimurium. Phage HK022, limm434, Limm434nin5 and limm434r32 are from the NIH or Columbia collections. (c) Plasmids Plasmid PJO210 is a pBR322 derivative expressing HK022 Nun (Oberto et al., 1989); plasmid pNAS200, a low copy number plasmid based on the incFI1 plasmid NRl, expresses I N from a plac promoter (Schauer et al., 1987). Plasmid pMK16 is TetR, KanR and is incompatible with pBR322 (a gift from D. Mills). Plasmids expressing Escherichia coli nusB or nusE genes were provided by D. Friedman, who also provided the following pBR322-based plasmids: pNAG15 (Am R TetS expressing E. COBnusA); pNAS1322 (AmpR Tet P expressing the nusA gene of S. typhimurium); pNAX1 (AmpR TetR expressing nusA hyl); pNAX2 (AmpR TetR expressing nusA hy2). The 5’ two-thirds of the nusA hyl gene is derived from E. coli and the remainder from S. typhimurium; the nusA hy2 gene is the reciprocal recombinant. Plasmid expressing rpoB and/or rpoC were obtained from A. Goldfarb and J. Lee. These include pGB218 expressing rpoB and rpoC (Barry et al., 1979), pXT7fl expressing rpoB (McKinney et al., 1987) and pT7fl expressing rpd: (Zalenskaya et al., 1990). The rpoC gene is cloned as an SphI-SspI fragment downstream from the phage T7 promoter. The fragment carries only 94 bpt of DNA upstream from rpoC including the weak P4 promoter (from which the gene is expressed) and a small portion of rpoB. The fragment terminates 144 bp distal to rpoC. (d) Enzyme assays Galactokinase and fl-galactosidase assays were performed as described (Robledo et al., 1990). (e) Amplijkation
of chromosomal DNA by PCR
A 1.6 kb E. coli chromosomal DNA fragment including nusAlO0 was amplified by the polymerase chain reaction (PCR) (Saiki et al., 1988). The amplification reaction was carried out for 30 cycles in the DNA Thermal Cycler (Perkin Elmer-Cetus) in a 100 ~1 final volume under the following conditions: the DNA was denatured at 94°C for 1 min, annealed at 60°C for 2 min with oligonucleotide primers (co-ordinates 795 to 819, 5’ to 3’, top strand; coordinates 2393 to 2417, 3’ to 5, bottom strand) and extended at 72°C for 3 min. To analyze the amplification product, a sample of the reaction mixture was subjected to electrophoresis on a 1% agarose gel and stained with ethidium bromide. The amplified DNA was purified by extraction with phenol and precipitation with ethanol, and analyzed by digestion with restriction endonuclease prior to cloning and sequencing. (f) Cloning the nusAlO0
mutation
The oligonucleotides used for amplification represented the nusA sequences described in section (e), above, and carried a 3’ tail bearing an EcoRI sequence. After purification, the amplified material was digested with EcoRI and t Abbreviations used: bp, base-pairs; PCR, polymerase chain reaction; kb, 10’ bases or base-pairs.
ligated into a lZapI1 vector predigested with EcoRI and dephosphorylated (from Stratagene Inc., La Jolla, CA). After packaging, white recombinant plaques were detected on IPTG/X-gal (isopropyl-/&n-thiogalactoside/ 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside) plates using strain XLl-Blue. Plasmid DNA was excised from the 1 clones in vivo, and isolated for sequencing (protocol from Stratagene Inc.). (g) DNA sequencing Double-stranded plasmid DNA was sequenced by the dideoxy method (Sanger et al., 1977), using Sequenase version 2.0 and following the protocol provided by the United States Biochemical Co. DNA was labeled with [“S]dATP at 1000 Ci/mmol (Amersham Inc.). Sequencing reactions were fractionated on 6% polyacrylgels, and run at a constant power of amide/7 M-Urea 70 W. After electrophoresis, the gels were fixed for 20 min with 10% (v/v) acetic acid, 12% (v/v) methanol. dried under vacuum for 1 h and autoradiographed at room temperature using Kodak XAR5 films.
3. Results (a) Isolation
and screening of Nun-resistant E. coli mutants
The nun gene product of HK022 blocks the expression of genes in the phage 1 pL and pR operons by terminating transcription at or near the n&L and n&R sites, respectively (see Fig. 1). E. coli strain R35 is an N99 derivative bearing a pR operon fusion to gal (from strain 6D7; Dambly-Chaudiere et al., 1983). The fusion has the structure: lcI857oRpR-cro-n&R-tRl-galETK R35 is Gall at 32”C,
(Fig. 1). since pR
is negatively
controlled by the ~1857 repressor. At 42”C, the temperature-sensitive repressor is denatured; the fusion is activated, and the strain becomes Gal+. The expression of gal is N-independent, since the efficiency of tR1 is approximately 50% (Robledo et al., 1990). In the presence of Nun, R35 is Gal- at all temperatures. By selecting from Gal+ colonies, we obtained mutants resistant to Nun termination. Mutations in nutR have been described (Robledo et al., 1990). Here we describe trans-acting mutations that identify
host factors
loci-t-N-nun-pL
required
~1657
for Nun activity.
pR-cro-nutR-tt?l-qoETK
(al n&R
CGCTCTTA cAcAl-T’ccA GCCC~AAGGGC
IWfL
CGCTClTA aAoATTa A GCCCTWAGGGC (b)
Figure 1. The 1 nutL and nutR fusions. (a) The 1 pL and I pR operons are shown linked to reporter genes. The direction of transcription is indicated by the broken arrows. The pL fusion carries uncharacterized weak terminators. The pR fusion includes the tR1 terminator, which, in its normal context, is -50% efficient. In strain N6028, which carries the pR fusion, the N gene is inactivated by amber mutations. The strains were described in detail by Robert et al. (1987). (b) The 1 nutR and I nutL Common nucleotides are sequences are compared. capitalized.
Nun Termination Spontaneous Gal+ derivatives of R35 carrying the Nun plasmid pJO210 (Oberto et al., 1989) were isolated on MacConkey galactose indicator plates at 42°C and screened for their ability to support the growth of ;limm434nin5. This hybrid phage carries the nut sites of L and is sensitive to Nun. The nin5 deletion removes the tR2 and tR3 terminators (Leason & Friedman, 1988), and allows the phage to grow in the absence of N, a pL operon function. In principle, therefore, a host mutation that blocked Nun termination at n&R would permit growth of limm434nin5. The screen yielded six independent mutants. To demonstrate the presence of active Nun in these mutants, we cured plasmid pJO210 by selecting transformants of the incompatible plasmid pMKl6, and then reintroduced pJO210. The resultant transformants remained Gal+, eliminating the possibility of a mutation in nun. A genetic analysis of the strains was then begun, to locate the mutant host genes. (b) Mapping
the host mutations
Since the nusA1, nusB5 and nusEY1 mutabions reduce Nun termination (Robert et al., 1987; Robledo et al., 1990), we first asked if our mutations were locat,ed in a nus gene. Using linked TnlO markers, we introduced the wild-type alleles of nusA, nusB, nusC (rpoB), nusD (rho) and nusE into the mutants by Pl transduction. The tetracyclineresistant transductants were then screened for their Gal phenotype and for sensitivity to superinfecting Aimm434nin5. Replacement of a mutant nus with a nus+ allele will yield a Gal- transductant resistant to kmm434nin5. By this means, we located one mutation each in nusA, nusB and nusE. Three mutations were mapped to the nusC region; they are, however, located in rpoC rather than rpoB (see below). The mutations were than moved into strain R35 lysogenic for HK022 by Pl cotransduction with the
Table 1 C’omplementation of host mutants Plasmid Host Wild-type nusAlO0 nusBlO0 nusEIO0 rpoCIO0 rpOCl01 rpoc102
None
nus+
o-3( +02) 7.3(+01) &0(+02) 47( f02) 64( f 1.0) 7.0( + 0.3) 7.7( 50.1)
@4( kO.3) @l( kO.1) @8( + @2) l.O(kO.1) l.l( kO.1) 1.3( kO.1)
in Phage HKOZZ
615
linked TnZO marker. Gal+ transductants were isolated for all six mutations, suggesting that each mutation represented a single genetic locus. The transductants were then transformed with plasmids expressing wild-type nus genes and the expression of galactokinase was determined. In the case of the putative nusA, nu.sB and nusE mutants, the wildtype gene, expressed from a multicopy plasmid, restored Nun activity (Table 1). As a control, mutants were also transformed with the other nus plasmids. The nusA mutant transformed with plasmids expressing nusB or nusE retained a mutant phenotype. Similarly, we saw no complementation when the nusB mutant was transformed with the nusA or nusE plasmids, or when the nusE mutant was transformed with nusA or nusB plasmids (data not shown). These mutants will be referred to as nusA100, nusBlO0 and nusE100. The nusC region mutations were suppressed by a plasmid (pT7j’; see Materials and Methods) that expresses rpoC (Table 1). The E. coZi DNA fragment cloned in pT7/I’ carries only 94 bp of DNA upstream and 144 bp downstream from rpoC; it is, therefore, unlikely to encode a gene in addition to rpoC. Plasmid pT7j did not suppress the nusAZ00 mutation, indicating that rpoC overexpression is not a general suppressor of nus mutations (data not shown). As shown by their Gal phenotypes, the nusC region mutations were also suppressed by pGB218, which expresses rpoB and rpoC, but not with pXT7/?, which expresses only rpoB (data not shown). These results indicate that the nusC region mutations lie in rpoC, rather than rpoB. We shall refer to these mutations as rpoClO0, rpoClO2 and rpoC102. To support further the map assignment of the we performed marker rescue rpoC mutations, experiments. The rpoC mutant strains bearing plasmid pT7/I’ are white (Gal-) on McConkey galactose indicator plates; rpoC+ expressed in multi-copy is dominant! to the chromosomal rpoC mutations. Incubation of these strains for two days at 42°C yielded red (Gal+) papillae at lOO-fold higher levels than the R35 HK022 lysogen. We presume that the Gal’ derivatives represent transfer of the rpoC mutations from the chromosome to the plasmid. This is the first evidence linking the /?’ subunit of RNP to Nun termination. (c) Effect of host mutations on I growth
Fusion: pR-cro-nutR-tRl-qalETK Values represent average galactokinase units from duplicate samples determined 1 h after shift from 32°C to 42°C (see Materials and Methods). Fluctuations are given in parentheses. The wild-type strain is R35 lysogenie for HK022; mutant alleles were introduced into the wild-type strain by PI transduction. The complementing nusA+, nusB+, nuaE+ and rpoC+ plasmids are described in Materials and Methods.
Classic E. cob nus mutations block N antitermination. As a consequence, 1 fails to propagate on nus mutants. To determine if our nus and rpoC mutations inhibited N antitermination, we plated &mm434 on the mutant strains at 42°C. The nusB100 mutation totally blocked phage growth. The nusElO0 mutation reduced the efficiency of limm434 plating. In contrast, the nusA100, rpoClO0, rpoClO1 and rpoClO2 mutants supported phage growth as well as wild-type strains (data not shown). Recall that all mutant strains were permissive for the N-independent &mm434 variant,
R. Robledo et al.
616
Table 2
Table 3
Activities of HK02.2 Nun and Lambda N at nutR
Activities of HK022 Nun and Lambda N at nutL
Plasmid Host Wild-type nusAIO0 nusBlO0 rpoclO0 rpoClO1 rpoC1OP
Plasmid
None
PN
pNun
45( f 0.9) 31( k9.1) 51(&@2) 3.9( 5 04) 44( 59.1) 57(+@6)
138( * 1.0) 1@5( + 94) 57( f05) 11.9( k 1.6) 123( +@8) 137( ko.2)
@7( kO.4) 7.2( f 0.6) 7.8( k @9) 6%( kO.3) &9( f 94) %9( * 0.7)
Fusion: pR-cro-nutR-tRl-galETK Galactokinase units were determined as for Table 1. Plasmid pN is pNAS299; plasmid pNun is pJO210. All strains are isogenic with R35 (wild-type).
limm434nin5. These results show that host mutations that block Nun termination do not necessarily also inhibit N antitermination. (d) Nun suppresses termination host mutants
at tR1 in
The nusA1, nusB5 and nusE71 mutations do not only abolish the termination activity of Nun, but invert Nun to a suppressor of the i tR1 terminator (Robledo et al., 1990). We show in Table 2 that the nusAl00 and rpoC mutations also reduce termination at tR1. In this experiment, we measured the galactokinase activity after induction of a pR-nutRtRl-galK chromosomal fusion. Termination at tR1 was as efficient as wild-type, approximately 50 o/o1in all mutant strains tested. Consistent with their phenotype for Iz growth, N antitermination at tR1 was unaffected by the nusA100 and rpoC mutations, but was blocked by the nusBlO0 mutation. The nusAlO0 mutation, the rpoC mutations, and possibly the nusBlO0 mutation, all inverted Nun to a weak suppressor of termination. (e) The host mutations are nut-speci$c Nun terminates transcription at or near the A nutR and the 1 nutL sequences (Robert et al., 1987). The two nut sequences are not perfectly homologous, and they differ in their locations relative to their cognate promoters and terminators. To determine if the host mutations also blocked Nun termination at nutL, we assayed fi-galactosidase expressed from a chromosomal pL-nutL-1acZ fusion (Table 3). In contrast to their effects at nutR, the mutations did not prevent Nun termination at nutL (Table 3, column 3), although the rpoClO1, rpoClO2 and possibly several of the other host mutations did reduce its efficiency. In the case of rpoClO2, the reduction in Nun termination has physiological consequences for the growth of A (see below). We found that the pL-nutL-ZacZ fusion was less active in all host mutations tested (Table 3, column 1). Perhaps the mutations increased the efficiency of weak termination sites within the fusion. N strongly
Host
None
PN
pNun
Wild-type nu.sAlOO nusBlO0 rp0C100 rpoClO1 rpoc1oe
1181 693 648 .554 574 441
4697 2837 653 3184 3483 3575
51 86 122 53 92 117
Fusion: pL-nutL-lad Values represent average fi-galactosidase units from duplicate samples determined 1 h after shift from 32°C to 42°C (see Materials and Methods). Fluctuation was 22% or less. All strains are isogenic with N6208 (wild-type).
stimulated the expression of ZacZ in wild-type hosts, and, with the exception of nusBlO0, in the mutant cells as well (Table 3, column 2; nusEZO0 was not assayed). (f) Why i is Nun-resistant in nusAlO0 and rpoC mutants The termination activity of Nun at nutL in the nusAlO0 and rpoC strains was surprising. The N gene lies in the pL operon just promoter-distal to n&L, and will not be expressed when Nun is active. N is essential for the growth of wild-type J. ‘and limm434. Why, then, is I Nun-resistant in these strains (see above)? The answer to this apparent paradox came from the analysis of the J. variant, Ar32. The r32 mutation consists of an IS2 insertion between tR2 and the 1 cII gene (Daniels et al., 1983). IS2 includes a strong rho-dependent terminator, which in the absence of N antitermination, completely prevents transcription of the downstream I 0 and P replication genes. Unlike wild-type A, IX?2 cannot replicate without N. Table 4 shows that 1r32 did not propagate on nusA100 and the rpoClO0 mutants in the presence of Nun (Table 4, column 2).
Table 4 Effect of host mutations on growth of I and lr32 Host Wild-type Wild-type/pNun nusAlO0 nwAlOO/pNun rpoC100 rpoClOO/pNun rpoClO2 rpoC102/pNun
I
h32
++ ++ ++ ++ ++ ++ ++
++ ++ ++ f-lf
1, -N-n&L-pL-cl-pR-cro-n&R-tRI-cII-0-P-tRP-tR3-Q ~~ PnutR-tRl-IS2-cII-O-P---1732, Phage are 2imm434. They were plated on wild-type (N99) or on isogenic mutant strains at 42°C. + + . normal growth; +, minute plaques; - , no growth.
Nun Termination The lr32 variant was partially resistant to Nun in rpoC102 mutants. Recall that the rpoC102 mutation reduced Nun termination efficiency in the pL operon (Table 3). We explain this result as follows. The nusA100 and rpoC mutations permit, wild-type 1 to express ,J genes 0 and P and to replicate in the presence of Nun. The replication of 1 results in the titration of Nun. N is then expressed, and N acts to antiterminate the distal tR2 and tR3 terminators in the nin region, allowing plaque formation. Since the Ar32 variant cannot replicate without N, Nun is not titrated, and the pL operon, including N, cannot be expressed. The growth of ir32 is blocked by termination at IS2. (R) The nusASa’ gene blocks Nun termination and N antitermination The existence of a nusA allele that specifically blocked Nun termination prompted us to examine the effe& of the Salmonella nusA gene on Nun termination in the pR and pL operons. NusASa’ does not support N antitermination (Friedman & Baron, 1974). We introduced nusAS”’ into E. coli by PI transduction and measured the expression of pRnutR-galK and pL-n&L-1acZ fusions. We found that the nusAS”’ allele blocked Nun inhibition and N stimulation of both the pR and pL fusions (Table 5). Tn addition, Nun stimulated the pR fusion suggesting that nusASa’ may also invert Nun into a weak suppressor of tR1. (h) Fine
m.apping of the nusAlO0 mutation
To map the nusA100 mutation, we first located the portion of the nusA gene required for Nun termination. Strains carrying hybrid E. colil Sal~monelln nusA genes have been constructed and tested for A plaque formation. A hybrid gene in which the 5’ two-thirds is derived from E. coli (hy-1) supports E, growth, whereas the reciprocal hybrid (hy-2) does not (A. Cranston, unpublished results,
Table 5 NusASa’ blocks both N antitermination Nun termination
and
617
in Phage HK022
1988). Two E. coli nusA cited by Friedman, mutations, nusA1 and nusAtsl1, lie in the 5’ twothirds of nusA: inhibition of both Nun termination and N antitermination by nusA 1 has been described (Robert et al., 1987; Robledo et al., 1990). To confirm the role of the amino-terminal portion of nusA in Nun termination, we introduced plasmids carrying nusAE, “‘li, nusAS”‘, nus4 hy-1 or nusA hy-2 into HK022 lysogens, and measured the activity of the pR-nutR-galK fusion. As expected, Nun blocked galK expression in strains bearing nusAE. “li or nusA hy-I, but not in nusASa’ or nusA hy-2 strains (data not shown). Assuming that the plasmid-born nusA alleles are dominant to the chromosomal nusA+, these results indicate that the 5’ two-thirds of the E. coli nusA protein is required for both N antitermination and Nun termination. (i) The sequence of nusA 100 As the first step in sequencing nusA100, the chromosomal allele was amplified by PCR and transferred to plasmid pBR322 (see Materials and Methods). Distance tests revealed that whichever nusA allele was present on pBR322, it was dominant for Nun termination (data not shown). This allowed us to locate the nusA100 mutation in a 778 bp AatII-NcoI fragment derived from the amino-terminal two-thirds of nusA. Replacement of the wild-type AatII-NcoI fragment with a fragment cloned from nusA100 yielded pBR322 clones conferring the mutant phenotype (data not shown). Conversely, substitution of the mutant fragment with wild-type converted a nusAlO0 plasmid clone into one with a wild-type phenotype. The sequence of nusA100 is shown in Table 6. We find a single base substitution, a G to A transition. The mutation converts codon 136 from glutamic acid to lysine. We note that the nusAlO0 mutation is located relatively far from the nusA1 (codon 183) and nusAtsl1 (codon 181) mutations and the arginine-rich domain (amino acids 170-I 91). In addition, we find four changes from the published nusA sequence (Ishii et al., 1984; Saito et al., 1986). These changes were detected independently by two groups (Craven & Friedman, 1991,
Table 6
Plasmid
Map of nusA mutations
Fusion
?&US A
None
PN
pNun
pR-nutR-galK pR-nutR-galK pL-n&L-lad pL-nutLlaci5
E. coli Sal E. coli ‘Sd
2.4( &@I) 2.3( + 63) 535 300
8.3( +@2) 3.5( f 0.2) 2156 229
@l(+O.l) 52( * 0.2) 21 305
Wild-type:
The pR and pL fusions are present in strains 6D7 and N6208, respectively. The nusA gene of S. typhimurium was introduced into both strains by PI transduction (see Materials and Methods). Values given in the top two lines represent galactokinase units as determined for Table 1; fluctuations are shown in parentheses. Reta-galactosidase units, shown in lines 3 and 4, were determined as for Table 3. Fluctuations in /?-galactosidase values were l3o/, or less.
AatIl 44
GAA Glu 136 ___
GM’ Gly 181 ___
(‘T(’
NC01
LeU
183 -
304
Mutants: AAA Lys nusAlO0
(:A(’ Asp nusAl1
( ‘G( ( k n~sA1
Codon sequences and amino acids of nusA mutations (bottom) compared to wild-type (top). Amino acid positions are counted from the translation start site of nusA. Restriction sites used for cloning are indicated.
618
R. Rob&o et al.
and Ito et al., 1991) and are in agreement with our sequence.
4. Discussion In this paper we report the isolation and characterization of E. coli mutations that block the activity of the phage HK022 Nun function. Nun provokes termination at or near the I n&R and nutL sequences. Our selection strategy demanded expression of gal from a JpR-nutR-tRl-gal operon fusion controlled by the lcI857 repressor. E. coli carrying this fusion are Gal- when Nun is active. Our selection for Gal+ colonies at 42°C yielded mutations in nusA, nusB and nusE, genes previously shown to play a role in both N antitermination and Nun termination (see Friedman & Gottesman, 1983; Friedman et al., 1984; Barik et al., 1987; Robert et al., 1987). In addition, we isolated three independent mutations in rpoC, the structural gene for the jl’ subunit of RNP. Unlike the well-documented role of the fl subunit of RNP (Jin et al., 1988), the involvement of the fl subunit in transcription termination has only recently been proposed. Evidence for this involvement is based on the isolation of rpoC suppressors of the rho201 (Jin & Gross, 1989) and the nusAtsl1 mutations (Ito et al., 1991). In addition, the 3’ nucleotide of in vitro paused transcripts can be cross-linked specifically to the p’ subunit (A. Goldfarb, personal communication). Our data implicate the /Y subunit in the termination reaction promoted by Nun factor. The rpoC mutations block Nun termination but not antitermination by 1 N function. In contrast, the rpoB mutation, nusC60, blocks N antitermination but not Nun termination (see Friedman & Gottesman, 1983; R.R., B.L.A. & M.E.G., unpublished results). The nusAlO0 mutation also specifically blocks Nun termination without affecting N antitermination. The 1 variant lr32, whose requirement for N antitermination is more stringent than wild-type A, grew normally in the nusAlO0 (and rpoC) mutants. Recall that the nusA1 mutation inhibits both N and Nun reactions (Robledo et al., 1990). Thus the elements of NusA needed for Nun termination and N antitermination are overlapping rather than identical. The nusA100 mutation represents a G to A transition at position 1260 resulting in a glutamic acid to lysine change at codon 136 within a revised nusA sequence. Like the nusA1 and nusAtsl1 mutations, nusAlO0 resides in the 5’ two-thirds of nusA. The nusAlO0 mutation lies 140 bp promoter-proximal to nusA1. Apart from its effect on Nun termination at nutR, nusAlO0 confers no detectable phenotype on its host. Our E. coli mutations also inverted Nun into a suppressor of tRI, an effect we have previously noted for the nusA1, nusB5 and nusE71 mutations, and for point mutations in the boxA sequence of nutR. We have yet to isolate a mutation that
confers a Nun null phenotype. Perhaps such mutants are temperature-sensitive to growth, and would, therefore, be eliminated in our selection protocol. We have selected host mutations that suppressed the Nun termination at nutR. When tested for Nun termination at nutL, the mutations were ineffective. The two nut sites differ in sequence, as do tL1 and tR1. The close location of nutR to tR1 is not duplicated at nutL. Which of these differences is relevant to the Nun phenotype is unknown. A selection based on suppression of Nun at nutL has not yet yielded bacterial mutants. Recall that the nusB5 and nusE71 mutations allowed 50% readthrough of nutL in the presence of Nun. The nusA1 mutation was less effective in blocking Nun termination, but still permitted significant readthrough (Robert et al., 1987). These nus mutations were selected as preventing N termination in both the pL and pR operons. Whether host mutations selected for N inactivation at nutL or nutR might be nut specific is an open question. We propose that after transcription of the I nut region, RNP, in concert with NusA, NusB and NusE, can be engaged by the I N or HK022 Nun gene product. Engagement by N produces an antitermination complex, whereas Nun engagement normally leads to rapid termination. Mutations in n&R boxA or in the Nus factors can produce a Nun complex capable of suppressing transcription termination at tR1. Possibly, the mutant Nun complex is short lived, since suppression of distal terminators is not observed (Robledo et al., 1990). Finally, and perhaps most importantly, our mutational analysis suggests that interactions between N and the b subunit are involved in formation of the antitermination complex, whereas Nun interactions with /I play a role in the termination complex formation. We thank U. Beauchamp for oligonucleotide preparations and for her help in sequencing the nusA gene. We are also grateful to Dr D. Friedman for various strains and plasmids, to Jookyung Lee for rpoB and rpoC plasmids, and to Dr R. Weisberg and S. Sullivan for criticisms and helpful discussions. This work was supported in part by NIH grant GM37219-03.
References Barik, S., Ghosh, B., Whalen, W., Lazinski, D. & Das. A. (1987). An antitermination protein engages the elongation transcription apparatus at a promoter-proximal recognition site. CeEl, 50, 885-899. Barry, G., Squires, C. L. & Squires, C. (1979). Control features within the rplJL-rpoBC transcription unit of Escherichia coli. Proc. Nat. Acad. Sci., U.S.A. 76. 4922-4926. Craven, M. G. t Friedman, D. I. (1991). Analysis of the Escherichia coEi nusAl0 (Cs) allele: relating nucleotide changes to phenotypes. J. BacterioE. 173, 1485-1491. Dambly-Chaudiere, C., Gottesman, M. E. Debouck, C. & Adhya, S. (1983). Regulation. of the pR operon of bacteriophage lambda. J. Mol. Appl. Cenet. 2, 45-56. Daniels, D. L., Schroeder, J. L., Szybalski, W., Sanger. F., Coulson. A. R., Hong, G. F., Hill, D. F..
Nun Termination Petersen, G. B. & Blattner, F. R. (1983). Complete annotated lambda sequence. In Lambda ZZ (Hendrix. R., Roberts, J., Stahl, F. t Weisberg, R., eds). pp. 519-676, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, KY. Friedman, D. I. (1988). Regulation of phage gene expression by termination and antitermination of transcription. In The Bacteriophages, vol. 2. pp. 263-319, Plenum Publishing Corp.. Rjew York. Friedman. D. I. & Baron, L. S. (1974). Genetic characterization of a bacterial locus involved in the activity of the X function of phage lambda. Virology, 58. 141- 148. Friedman, 11. I. & Gottesman, M. E. (1983). Lytic mode of lambda development. In Lambda ZZ, pp. 21-51. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, SY. Friedman, 11.I.. Olson, E. R., Georgopoulos, C.. Tilly. K.. Herskowitz. I. & Banuett. F. (1984). Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbial Rev. 48. 299-325. Ishii. S.. Ihara. M.. Maekawa, T., Xakamura, Y., Uchida. H. & Imamoto, F. (1984). The nucleotide sequence of the cloned nusA gene and its flanking region of Escherichia roli. Nucl. Acids Res. 12, 3333-3342. Ito. K., Egawa~ K. & Xakamura, Y. (1991). Genetic interaction between the /?’ subunit of RNA polomerase and the arginine-rich domain of EscherichLa coli nusA protein. J. Bacterial. 173, 1492-1501. Jin, D. J. & Gross. (1. A. (1989). Three rpoBC mutations that suppress the termination defects of rho mutants also affect the functions of nusA mutants. Mol. Gen. &net. 216, 269-275. Jin, I). ,J.. Walter, W. A. & Gross, C. A. (1988). Charact,erization of the termination phenotypes of refampicin-resistant mutants. J. Mol. Biol. 202, 245-253. Lazinski, I).. Grzadzielska, E. & Das, A. (1989). Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell, 59, 207-218. Leason. K. R. dz Friedman, D. 1. (1988). Analysis of transcription termination signals in the nin region of bacteriophage lambda: the rot deletion. J. Bacterial. 170. 505lm--5058. McKinney. .J. D.. Lee, J., O’Neill, R. E. 6 Goldfarb, A. (1987). Overexpression and purification of a biologicall3 active rifampicin-resistant /I subunit of Eacherichia coli RPU’A polymerase. Gene, 58, 13-18.
in Phuge HK022
619
Oberto, J., Weisberg, R. A. & Gottesman, M. E. (1989). Structure and function of the nun gene and the immunity region of the lambdoid phage HK022. J. Mol. Biol. 207, 675-693. Olson, E., Tomich, C. & Friedman, D. I. (1984). The nusA recognition site. Alteration in its sequence or position relative to upstream translation interferes with the action of the X antitermination function of phage lambda. J. Mol. Biol. 180, 1053-1063. Robert, J., Sloan, S. B., Weisberg, R. A., Gottesman, M. E., Robledo, R. & Harbrecht, 1). (1987). The remarkable specificity of a new transcription termination factor suggests that the mechanism of termination and antitermination are similar. Call, 51, 483-492. Robledo, R., Gottesman, M. E. & Weisberg, R. A. (1990). 1 nut R mutations convert HK022 Xun protein from a transcription termination factor to a suppressor of termination. J. Mol. Biol. 212, 635-643. Saiki, R. K., Gelfand, D. H., Stoffel, S.. Scharf, S. J.. Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988). Primer-directed enzymatic ampliiication of DNA with a thermostable DNA polymerase. Science, 239, 487-491. Saito, M., Tsugawa, A., Egawa, K. & Xakamura, Y. (1986). Revised sequence of the nusA gene of Escherichia coli and identification of nusA I1 (ts) and nusA1 mutations which cause changes in a hydrophobic amino acid cluster. Mol. C&n. Genet. 205. 380-382. Sambrook, J. F., Fritsch, E. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. Sanger. F.. Nicklen, S. & Coulson, A. R. (1977). DSA sequencing with chain-terminating inhibitors. Proc. Nat. A cad. Sci., U.S. A. 74, 5463-5467. Schauer, A. T., Carver, D. L., Bigelow, B., Baron, L. 6. & Friedman, D. I. (1987). 11v antitermination system: functional analysis of phage interactions with the host NusA protein. J. Mol. Biol. 194, 679-690. Silhavy, T. J., Berman, M. L. & Enquist. L. W. (1984). Experiments in Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Zalenskaya, K., Lee, J., Gujuluva, C. X.. Shin, Y. K., Slutsky, M. & Goldfarb, A. (1990). Recombinant RNA polymerase: inducible overexpression, purification and assembly of Esch,erichia coli rpo gene products. Gene, 89, 7-12.
Edited by P. von Hippel
