Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli K-12

J. Mol.

Hid.

(1988)

203,

15-27

Mutations in rpoD that Increase Expression of Genes in the ma1 Regulon of Escherichia coli K-12 James C. Hu-f and Carol

A. Gross

Department of Bacteriology University of Wiscon,sin, Madison, WI 53706, U.X.A. (Received

11 August

1987)

The o subunits of eubacterial RNA polymerases determine the site selectivity of initiation of transcription at promoters. Mutations in rpoD, the gene that encodes 070, the major 0 factor in Escherichia coli, should be useful in determining the molecular details of the process of transcription initiation. However, such mutations are likely to be deleterious or lethal, since 07’ is an essential gene product. We designed a system for the rapid isolation and fine structure mapping of mut,ations in rpoD, which allows selection of mutations that would otherwise be deleterious to the cell. WC used this system to isolate a new class of mutations in rpoD, mutations that relieve the requirement, for CAP-CAMP for initiation at promoters in t,he ma1 regulon. These mutations, which we designate rpoD(Ma1) mutations, occur in two clusters in t’he rpoD gene within regions previously suggested by amino acid sequence comparisons to be important for rs structure or function. We cannot distinguish whether the rpoD(Ma1) mutations affect ma1 expression b.y altering interaction between RNA polymerase and ma1 promoters or between RNA polymerase and the accessory transcription factor MalT. However, the effects of the mutations on activator-independent transcription from the lac promoter (4 rpoD(Ma1) mutations decrease CAP-independent expression of the lac promoter in rive) suggest that the regions of cr ident’ified by our mutations may be directly involved in promoter recognition.

1. Introduction

Ea7’ are likely to be deleterious. Such mutations are also likely to be rare. To overcome these problems, we have developed a system that allows us to localize mutagenesis to a phasmid-encoded copy of rpoD, which can be maintained in a host containing the wild-type rpoD gene. We used this system to isolate a new class of mutations in rpoD, mutations that relieve the requirement for CAPCAMP for initiation at promoters in the ‘ma1 regulon, which we call rpoD(Ma1) mutations. The ma1 regulon of E. coli consists of t#hree operons that contain genes required for maltose utilization, malP&, malEFG’ and m&K-la.mR. Expression of the ma1 regulon is controlled by both CAP-CAMP (Perlman & Pastan, 1969; GuidiRontani & Gicquel-Sanzey, 1981) and the malT gene product, a regulon-specific activat,or (Schwartz, 1967; Hofnung et al., 1971). CAPcAMP regulates wzal regulon expression at two different levels; it is required for efficient expression of malT (Debarbouille & Schwartz, 1979) and directly stimulates transcription of two of t,he ma1 promoters, PmolE and PmalK (Chapon, 1982a). By selecting for CAP-independent growth on malt,ose,

Bacterial RNA polymerase is found in two forms. Core RNA polymerase (E) catalyzes the DNAdependent synthesis of RNA, but does not initiate transcription at specific sites. The r~ subunits of bacterial RNA polymerases are required for the selective initiation of transcription by RNA polymerase holoenzyme (Ecr) (Burgess & Travers, 1970; Reznikoff et aZ., 1985). The detailed mechanistic role of fr factors in promoter selection is not known. In order to advance our understanding of how cr directs RNA polymerase to initiate at, promoters. we have selected and characterized mutant cr factors that, affect transcription initiation by their cognate forms of RNA polymerase holoenzyme. In Escherichia coli, the major form of 0 is a 70,263 &IF protein encoded by the rpoD gene. Mutations that) affect the initiation specificity of t Present address: Department of Biology, Massachusset,ts Tnstitute of Technology, Cambridge, 02139,

MA

IJ.S.A.

OW-2836/8X/

I7001 5-l 3 $03.00/O

15

J. C. Hu and C. A. Gross

16

we can isolate mutations in rpoD that affect initiation at the ma1 promoters; these mutations can then be characterized for other effects on gene expression. In this paper, we describe the isolation, intragenie mapping and characterization of rpoD(Ma1) mutations. These mutations occur in two clusters in the rpoD gene, corresponding to two regions of conserved amino acid sequence found in several (T factors.

2. Materials

and Methods

(a) Media and strain constructions and M9 plates and liquid media, and MacConkey medium were made as described by Miller (1972). M9 plates contained sugars at 0.2 y0 (w/v) and amino acids at 20 fig/ml. Kanamycin (Kan, 30 pg/ml), ampicillin (Amp, 100 pg/ml) and tetracycline (Tet, 20 pg/ml) were added sterilely as indicated. Transductions using Pl vir were done aa described by Miller (1972). Bacterial strains used in this study are listed in Table 1. Transductions to Acya were done in 2 steps: first, transduction to Acya by screening for LB agar

cotransduction with ilv: :Tn5, which is approximately 50% linked to cya. Second, transduction of the resultant Acya strain to Ilvf using Pl vir grown on a known Acya ilv+ donor. Intermediates in these constructions have been omitted from Table 1. (b) Phmmid

construction

Construction of pJH62, a plasmid that carries the intact ~7 operon, is illustrated by Fig. 1 and described in the legend. The phasmid lpJH62 was constructed by cloning the plasmid pJH62, linearized with EcoRI, between the EcoRI sites of Xh25 (Gross et al., 1979). Ligated DNA was transfected into MC1061 and plaques were screened for the presence of kanamycin-resistant (Kan’) lysogens. Spontaneously released phage from such lysogens were plaque-purified and grown by standard 1 techniques (Arber et al., 1983).

RNA was prepared from cells growing exponentially in M9 glucose medium by the method of Jinks-Robertson et al. (1983).RNA wasprecipitated with ethanol repeatedly from 2 M-ammonium acetate to remove salts carried over from the culture medium. For S, mapping experiments,

Table 1 Strains Strain A. For piaemid MC1061

Genotype and phage construction F- araD139 A(araleu)7697 Ala&74 galU galK hsdR &A

B. For mutagenesis, KD1067 pop3937 GAG4222 CAG7186

C. For transfer GAG4336 CAG4347

pop3122 pop3125 CAG4310 CAG43 11 CAG4312 pop3955 CAG4384 GAG4274 CAG7905 CAG7906 GAG7907 CAG7908 CAG7985 CAG7986 CAG7987

selections and mapping F- muiD arg his F- araDi39 (&cPOZ YA)U169 rpsL relA jlbB malTpi’ pop3937 Acya (limm2’) F- C%OOK-[thi leu thr lac Y tonA eupE44 galK] Acya, (&mm21 redd)

to the chromosome GAG4222 dnaG3-TnlO pop3669 malTp7 Acya (limm2’) dnaG3-TnlOKan

D. For measurements pop3969

used in this work Source

M. Casadaban

M. Howe C. Chapon This study D. Siegele

This study This study

of effects wn gene expression F- araD139 (AlucPOZYA)U169 rpsL relA flbB &malK-IacZ) F- araD139 (AlacPOZYA)U169 rpaL reEA $bB &nuzlE-ZacZ) F- araD139 (AlacPOZYA)U169 rpaL relA jlbB &malP-lucZ) GAG4222 lpop3069 CAG4222 lpop3122 CAG4222 Apop3125 malTp7: :lacZ pop3955 Acya CAG4222 araBA: :MudX4064 pop3937 (Iplac5) rpoD+ zgh: :TnlO pop3937 @p&5) rpoD820 zgh: :TnlO pop3937 (rlplac5) rpoD821 zgh: :TnlO pop3937 (Iplac5) rpoD835 zgh: :TnlO pop3937 (I&5) dpJH62rpoD+ pop3937 @p&x5) lpJH62rpoD823 pop3937 (Iplac5) lpJH62rpoD824

M. Hofnung M. Hofnung M. Hofnung This study This study This study M. Schwartz This study D. Siegele D. Siegele D. Siegele D. Siegele D. Siegele D. Siegele D. Siegele

rpoD

Muhtions that Alter Gene Expression

50 pg of total cellular RNA was hybridized to 1 pg of pSD13 cleaved and 5’-end-labeled at the BamHI site. Hybridization and S, cleavage were done as described by Berk & Sharp (1977). Protected fragments were run on a 6% (w/v) polyacrylamide gel containing 6 M-urea. Bands were located by autoradiography, and quantified by cutting the bands out of the gel and measuring Cerenkov radiation. (d) Mutagenesis lpJH62 was grown on KD1067, which contains the mutD5 mutator allele, to make mutagenized phasmid stocks, or on control mutD+ strains. Mutator activity was induced by growing KD1067 in the presence of 5 pg thymidine/ml (Fowler et al., 1974). Phasmids forming clear plaques were present in mutagenized stocks at approximately 1%. Independent lysates were used to isolate each mutant. Mutagenized and control phasmid stocks were used to infect CAG4222: 0.1 ml of infected cells was mixed with 1 ml of LB and preincubated for 1 h to allow expression of kanamycin resistance, pelleted by centrifugation, resuspended in MS salts, and plated in parallel on M9 maltose plates containing kanamycin, and on LB plates containing kanamycin to determine the number of Kan’ lysogens. Mal+ candidates were colony-purified, and grown in liquid culture in NZY broth. Released phasmids were recovered from the culture supernatant, residual bacteria were killed with chloroform, and the phasmid stocks were tested for their ability to transfer the Mal+ phenotype by infecting a fresh culture and spotting a drop of the infected cells on MacConkey maltose plates containing kanamycin. Similar spot tests were used to determine whether the mutant phasmids could lysogenize cells containing either a temperature-sensitive (ts) mutant cr encoded by the rpoD800 allele (Liebke et al., 1980) or a nonsense mutation, rpoD40 (Osawa t Yura, 1980) in the presence of a ts nonsense suppressor, SupliR”. (e) Mapping

mutations

within

rpoD

7 Abbreviations used: kb, lo3 bases or base-pairs; wild-type; ts, temperature sensitive.

select for phage particles that carry DNA from both the Kan’ phaamid and the Amp’ mapping plasmid. Amp’ , Kan’ lysogens were screened for the fraction of rpoD+ recombinants. (r) Subcloning

wt,

and DNA sequencing

To determine the DNA sequence of the mutations, we moved the mutant Q alleles from the phasmids to smaller plasmid subclones. We subcloned the viable mutations by cleaving the mutant phasmid DNA with EcoRI and religating to regenerate pJH62 carrying the mutant alleles. Mutant phasmids were purified from IO-ml liquid lysates by centrifugation. Phasmid DNA was purified by extraction with phenol and precipitation with ethanol. Purified phasmid DNAs carrying the rpoD820, rpoD821 and rpoD833-842 alleles were cleaved with endonuclease EcoRI (to remove Charon 25 DNA), ligated and used to transform competent E. coli MC1061 to Kan’. We expected that some of the non-viable mutations might be deleterious if expressed from higher copy number plasmids. We subcloned these alleles in constructions that do not express functional a70. Phasmids carrying the rpoD822 to rpoD832 alleles were cleaved with a mixture of EcoRI and BumHI (to remove Charon 25 DNA along with r~ operon DNA upstream from the BumHI site in rpoD) followed by treatment with DNA polymerase I large fragment in the presence of dNTPs to fill in overhanging ends. The digests were then ligated to generate a plasmid containing rpoD sequences downstream from the BumHI site, equivalent to a deletion between the EcoRI and BamHI sites of pJH62. Plasmid DNA was prepared by precipitations with polyethylene glycol as described by Humphreys et al. (1975). End-labeled restriction fragments were prepared as described by Maxam t Gilbert (1980). DNA sequencing was done either by the chemical cleavage method of Maxam & Gilbert (1980) or by the ( - ) reaction dideoxy method of Seif et ~2. (1980). (g) Transfer

Mutations on the phasmid were mapped against a set of “mapping plasmids” bearing deletions of varying amounts of the N terminus of the rpoD gene (Hu & Gross, 1983). Mutant phasmid stocks were prepared from culture supernatants of mutant candidates grown overnight in NZY medium. These stocks contained a mixture of phasmids and &mm” red+ phage released by CAG4222. Phasmids in these stocks were used to lysogenize CAG7186 (C600 galK, Acya, @mm” red3)). This purifies the mutant phasmids away from contaminating Ired+ phage, which can raise the background frequency of recombination with the chromosome. Supernatants from CAG7186 lysogens, containing a mixture of released phasmid and the red3 resident prophage, were used to prepare plate lysates on strains containing mapping plasmids. Plate lysates were also prepared on cells containing the plasmid pDAS50, which contains about 1 kbt of DNA downstream from the o operon but lacks rpoD sequences, for negative controls. These lysates were then used to infect the indicator strain CAG4222. Infected cells were plated on MacConkey maltose indicator plates containing kanamycin and ampicillin to

17

of rpoD mutations from the phasmid to the chromosome

Pl vir was grown on lysogens containing the mutant phasmids in CAG4336 (CAG4222 dnaG3 zgh: :TnlO). These Pl stocks were used to transduce CAG4347 (pop 3669 mulTp7 Acya dnuG3 zgh : : TnlOKan) to simultaneous tetracycline resistance and temperature resistance (dnaG+ ) at 37 “C on plates containing X-gal as an indicator of the activity of the m&K: :EacZ fusion in this strain. Blue, Tet’ transductants were screened for kanamycin sensitivity, which showed that the phasmid was not transferred. Kar? candidates were then screened for the ability to donate the mutant phenotype by contransduction with zgh: :TnlO. (h) Effects on gene expression For the viable mutations, rpoD820, rpoD835 and rpoD821, expression in cells haploid for the mutant rpD alleles was compared to expression in isogenic rpoD+ strains. The 2 non-viable alleles rpoD823 and rpoD824 were introduced on phasmids into cells with a wt copy of rpoD on the chromosome. Gene expression in these strains was compared to isogenic strains carrying an rpoD+ phasmid. pop3069, pop3122 and ~0~3125, containing 1acZ fusions to the malK, m&E and m&P genes, respectively,

18

J. C. Hu and C. A. Gross

were generously provided by M. Hoffnung. Specialized transducing phage carrying lucZ fusions were isolated from these strains by ultraviolet light induction, plaquepurified and grown. These phage were then used to lysogenize CAG4222. Lysogens were screened for maltoseinducible /I-galactosidase expression. Transduction experiments with n&A: :TnlO showed that the fusion phages had integrated by homologous recombination with the resident imm21 lysogen. rpoD(Ma1) mutations were introduced into these strains by Pl transduction for viable alleles or by lysogenization with mutant phasmids for non-viable alleles. Expression from mal promoters was measured by measuring steady-state /I-galactosidase synthesis m a differential rate (Ingraham et al., 1983). B-Gelactosidaae activity was assayed at 4 to 5 points during exponential growth in M9 minimal medium containing glucose at 37 “C!. The data were fit to a straight line. /I-Galactosidase activity is expressed as Miller units (Miller, 1972). Kanamycin was added to cultures of phasmid lysogens. pop3955, containing a malT: :EacZ protein fusion linked to wmZTp7 (Chapon, 19825) wa.q kindly provided by M. Schwartz. rpoD(Ma1) mutations were introduced into CAG4384, a Acya derivative of pop3955, by Pl transduction or by lysogenization with mutant phasmids. The mutant phwmids were introduced into CAG4384, which is I-resistant, by phenotypic mixing with @89. fi-Galactosidase synthesis from CAG4384 derivatives carrying rpoD(Ma1) mutations was measured in cells growing exponentially in M9 glucose containing amino acids at 37°C as described above for the other ma1 fusions. Expression of araBAD was measured using an araBA : : ZucZ fusion derived from a Mu dX insertion described previously (Hu & Gross, 1985). This fusion was moved into CAG4222 by transduction with Pl vir by selecting for the Mu dX-encoded ampicillin resistance and screening for arabinose inducibility of fl-galactosidase activity and linkage of the insertion to leu. rpoD(Ma1)

mutations were introduced by Pl phasmid lysogenization measured in cultures

Tn5 (Fig. 1). We cloned the entire plasmid into Charon 25 (Gross et al., 1979), a 1 vector with the immunity region of phage 21. The resultant phasmid, IpJH62, has a number of useful pro erties. It grows lytically to high titer (10’ to 10’ r plaque-forming units/ml). The presence of the ColEl replication functions allows infecting phasmids to establish Kan’ lysogens in homoimmune (imm21) hosts at a frequency of approximately 25% (data not shown). Kanr lysogens stably maintain the phasmid; fewer than 1 y. (l/270) of cells grown in the absence of selection lose resistance to kanamycin. Phasmid stocks of lo6 plaque-forming units/ml can be recovered from the culture supernatants of lysogens. To measure the fraction of total a7’ transcripts originating from the phasmid in lysogens, we compared rpoD transcription from the chromosome and the phasmid in cells lysogenic for lpJH62. We genetically marked the chromosomal copy of rpoD by introducing the rpoD800 allele into the chromosome of a phasmid-containing strain. Since this allele is a 42 base-pair intragenic deletion in rpoD, fragments of different sizes are protected from S, digestion by the chromosomal and phasmid-derived mRNAs, as shown in Figure 2. Quantification of these protected fragments showed that five- to sixfold more rpoD mRNA is transcribed from the phasmid than from the chromosome, suggesting that cells contain five to six copies of the phasmid. The level of a70, as determined by Western blotting, in phasmid-containing cells is about threefold higher than in cells haploid for rpoD (data not shown).

transduction or

(b) Mutagenesis and selection pseudorevertants of Acya

and expression of the fusion was growing exponentially in MS

minimal medium containing glucoseas a carbon source and 2% (w/v) arabinose as an inducer. lac expression was measured in strains containing the luc transducing phage IpZac5. rpoD(Mel) mutations were introduced by Pl transduction or phasmid lysogenization and /I-galactoeidase activity measured in cultures growing exponentially in M9 glucose medium with 1 mMisopropyl-thio-b-n-galactoside to induce lox operon

expression.

3. Results (a) Construction and characterization of a phasmid vector carrying the o operon To localize mutagenesis to rpoD, and to select mutations that might be otherwise deleterious to the cell, we designed the phasmid ApJH62. The presence of a wt chromosomal copy of rpoD in phasmid-containing strains should allow cells to grow even with mutant phasmids that carry rpoD alleles that do not support normal transcription. The plasmid pJH62 contains the 0 operon in a ColEl vector, pRZ112 (Jorgensen et aE., 1979), which also carries the kanamycin resistance gene of

of Mal+ mutants

The previously described rpoD(Alt) mutations allow cells defective for CAP-CAMP to grow on arabinose, but do not allow them to grow on maltose (even in the presence of malTp7; see below); in fact, ma1 operon expression in rpoD(Alt) strains is lower than in rpoD+ cells (Hu & Gross, 1985). Thus, mutations in rpoD that increase ma1 expression should represent a new class of 0 mutants. In the absence of CAP-CAMP, ma1 expression is virtually nonexistent; mutations that affect the affinity of RNA polymerase for ma1 operon promoters might not increase mu1 expression enough to allow growth on maltose. To allow selection of mutations with relatively small effects on ma1 expression, we used strains containing malTp7 (Chapon, 1982b). This mutation increases ma1 expression by raising the basal level of m&T expression, but does not allow CAMP-independent growth on maltose. We mutagenized ApJH62 by pastssage through a mutD5 strain (seeMaterials and Methods), infected CAG4222, a &mm21 lysogen that carries Acya and malTp7, and selected Mal’ colonies. Malf mutants were found 10 to 1999 times more frequently in CAG4222 lysogenized with mutagenized lpJH62 than

in

cells

lysogenized

with

unmutagenized

rpoD Mutations

that Alter Gene Expression

19

BumHI

BumHI

(a)Add HindIU linkers to Pvull. ~i%?ltiii into pRZ112

BornHI

clone

(b)Clone fragment

1 .EcoRI

f

pJH61

EcoRl

C/al

_.

J

EcoRI

(c)Clone EcoRI-BumHI fragment from pJH60 into pJH61, regenerating intact operon

Chl-6’sormHI into pBR322

dnaG pJH62

BumHI

L

Hind

IlI

1

BarnHI

Figure 1. Construction of pJH62. A complete copy of the CToperon was constructed in several steps in pRZ112, a derivative of ColEl: :Tn5 (Jorgensonetal., 1979)tc generatethe plasmidpJH62. (a) Hind111 linkers were ligated to the ends of a 3 kb PvuII fragment from pMRG1, which contains rpoD DNA downstreamfrom the codon for the 8th amino acid of a76 and downstream flanking sequences. This fragment was cloned into the Hind111 site of pRZl12. Clones were picked in which the direction of rpoD transcription was clockwise, as shown in the Figure. One such clone was named pJH61. (b) A CZaI-BarnHI fragment containing the Q operon promoters, the dnoQ gene, and the N-terminal portion of rpoD was cloned between the CZuI and BumHI sites of pBR322 to generate pJH66. (c) The EcoRI-BarnHI fragment containing upstream operon sequences from pJH66 was cloned between the EcoRI and RumHI sites of pJH61 to form pJH62, which contains an intact a operon.

phasmids (Table 2). Mal+ revertants on the mutagenized plates were visible earlier than on control plates, indicating that revertants in the control plates were arising from a lawn of background growth. Greater than 85% (20/23) of the revertants from the mutagenized infection transferred the Mal+ phenotype with the spontaneously released phage. The frequency of Mal’ mutants varied with the temperature of the selection; lOO-fold fewer Mal+ colonies were observed at 42°C than at 30°C. Twenty-three independent Malf revertants, or rpoD(Ma1) mutations, were isolated. Thirteen of these, rpoD821 to rpoD833, were selected at 42°C.

The other ten, rpoD820 and rpoD834 to rpoD842, were selected at 30°C. Four of the isolates selected at 30°C (rpoD834, rpoB836, rpoD837 and rpoD839) were mucoid. (c) Temperature dependenceof the Ma1 phenotype The difference in recovery of Mal+ mutants at 30°C and 42 “C led us to test the temperature dependence of the Mal+ phenotypes of the mutants. All of the mutants isolated at 42°C were Mal+ at 37°C and, with one exception, at 30°C. The

20

J. C. Hu and C. A. Gross

Table 2 BonHI

Ejkiency of mutagenesis

800nt rpoD + RNA

400 nt

*

b promted

Mutagenesis lmgment

.AWQ800 r~oD800

RNA

I

b

300 nt l

promdud

Corracted oltandsinlure3

(bItI

fragment

ctr/min

mutD5 None mutD5 None

Temperature ( “C)

Mal+ Kan’

30

O/c transfer

lysogens

w/phmmid

5 x 1o-4
42

npJH62 was mutagenized described in the text. n.d., not determined.

mutants/

and

used

to infect

> 85 y. n.d.

CAG4222

a8

37°C. The difference in frequency of Mal+ mutants selected at 30°C and 42°C reflects the fact that many more mutational changes lead to the Mal+ phenotype at the lower temperature.

rpoLJ800

-+

200c?l/min

Figure 2. rpoD expression from the phasmid lpJH62. (a) Schematic of the experiment. The 32P-labeled DNA probe was prepared from the plasmid pSD13 (Hu & Gross, 1983)5’ end-labeled (indicated by an asterisk) at the BamHl site in rpoD and recut with EcoRI. DNA removed by ASD13 is indicated by the open box. RNA from wt rpoD and rpoD800, shown as arrows, differ in sequence due to the presence of the 42 base-pair rpoD800 deletion, indicated by the open box in the lower RNA. Hybrids between the pSD13 probe and RNA from each allele can be distinguished by the size of the fragment protected against S, nuclease digestion as indicated. rpoD+ RNA protects about 466 nucleotides (nt) of the probe, from the BamHI end to the endpoint of ASD13. rpoD800 RNA protects about 366 nucleotides of the probe, from BumHI to the endpoint of ArpoD800. The undigested probe runs at 806 nucleotides, and is thus distinguishable from both types of protected fragments. (b) Autoradiogram showing DNA protected against S, digestion by RNA prepared from cells containing rpoD+ , rpoD800, or both alleles. Lane 1 shows no protection of the probe by the tRNA control. DNA protected by RNA from CAG4225 (chromosomal rpoD800, lane 2) and CAG4223 (chromosomal rpoD+, lane 4) were included as size standards. Lane 3 shows protection by RNA from CAG4337 (lpJH62--rpoDC/rpoD800). Relative expression of rpoD mRNA from the chromosomeand the phasmidin GAG4337were determined as rpoD+/rpoD800 cts/min in lane 3. Cts/min from the bands in lane 3 are shown adjacent to the autoradiograms; these numbers are corrected for background by subtracting the average cts/min in gel slices taken above and below each band. Similar ratios were determined by scanning denaitometry (data not shown). exception was rpoD823, which failed to grow at 30°C. In contrast, nine out of ten mutants isolated at 30% were Mal- at 42”C, and three of these, rpoD834, rpoD836 and rpoD837, were Mal- at

(d) Do the o mutants support growth in the absenceof wild-type 0701 The system described above was designed to allow selection of mutations that do not allow growth in normal, haploid cells. To ask whether the mutant o factors could support cell growth, we tested their ability to complement known conditional lethal mutations in rpoD. We introduced the mutant phasmids into cells carrying rpoD800, which encodes a ts cr polypeptide, or cells carrying the amber mutation rpoD40, and a ts suppressor tRNA. Cells were scored for their ability to grow at the restrictive temperature (42°C). All of the cr alleles selected at 30°C and one of the mutants isolated at 42°C complemented both rpoD40 and rpoD800. We refer to these as viable alleles. However, we were unable to introduce 12 out of 13 of the mutant isolates selected at 42 “C into cells containing either defective chromosomal rpoD allele at any temperature. In fact, one of the 12, rpoD823, causes cells to be cold-sensitive even with the wt rpoD allele present on the chromosome. We refer to these rpoD(Ma1) alleles as non-viable alleles. (e) Intragenic mapping and sequencing the rpoD( Mal) mutations We used a set of multicopy plasmids carrying the 3’ end of rpoD and deletions of varying amounts of the 5’ end of the gene (see Fig. 3) to map the rpoB(Ma1) mutants carried on the phasmid. Recombination between the phasmid and the mapping plasmid during lytic growth generates cointegrate structures that are small enough to be packaged by phage 1. These cointegrates are selectable, as they carry antibiotic resistance markers from both the phasmid and the mapping plasmid (Fig. 3). Deletion plasmids with DNA homologous to rpoD on both sides of the mutation lead to two classesof cointegrates: crossovers downstream from the mutation retain the mutant allele, while crossovers

rpoD Mutations

A +JHE~

J

rpoD

21

that Alter Gene Expression

-

+

OSR Kan

Amp

‘tpoD(Nwm?ina/

’ rpoD I pJH62

A)

(linked

to muration) Karl

Amp

Figure 3. Mapping

scheme. Phaamids containing mutant rpoD alleles (indicated by heavy lines) were grown on cells carrying plasmids with varying amounts of wild-type rpoD DNA (shaded lines). Recombinants that integrated the deletion plaamid into the phaamid were selected by demanding simultaneous resistance to ampicillin and kanamycin. lysogen. Recombination to the left of the Recombinants with both drug resistance markers arose at lo-’ to 10-4/Kan’ mutation (as shown) will generate a cointegrate with an intact rpoD+ allele and a deleted gene containing the mutation, while recombination to the rieht of the mutation Inot shown) will generate a cointegrate that retains an intact mutant rpoD

allele

and a deletedgenecontaining rpoD+. ’

upstream from the mutation replace mutant DNA with wild-type sequences (Fig. 3). Thus, wild-type (rpoD+) recombinants among the selected cointegrate population indicate that the deletion plasmid carries DNA covering the mutation. In contrast, if DNA that covers the mutation is deleted from the plasmid, only cointegrates expressing the mutant gene will be formed, since recombination can only occur downstream from the mutation. Recombination between the cointegrates and the chromosome can regenerate a wt rpoD gene. This background is determined by measuring the fraction of wt recombinants in crosses with a plasmid carrying only DNA downstream from rpoD. Table 3 shows mapping results for two mutations. Mutant and wt recombinants were distinguished by screening for the mutant phenotype as determined by their color on MacConkey maltose indicator plates. Thus, the mutants were mapped as lying to the right of particular deletion endpoints by determining the smallest plasmid to give a number of white colonies significantly over background. Data of this kind placed the rpoD(Ma1) mutations in two clusters within the rpoD gene. We refer to these as the upstream cluster (actually well within the C-terminal half of rpoD) and the downstream cluster. All of the non-viable mutations mapped to the downstream cluster, to the right of our last deletion endpoint, a ClaI site about 180 base-pairs from the C-terminal end of rpoD. DNA sequencing identified mutational changes for 15 of the rpoD(Ma1) mutations. We found three different sequence changes in the upstream cluster and three different changes in the downstream cluster. All 12 of the non-viable isolates were found to be one of two different sequence changes in a

single arginine codon at position 584. The identities and the positions of the mutations are shown in Figure 4. Properties of the rpoD(Ma1) mutants are summarized in Table 4. Five alleles with different sequence changes were used in the experiments described below. The amino acid change and position

rather

than

the allele

number

will

be used

to identify each mutation, for example, the Ala to Val change at position 415 caused by rpoD820 will Table 3 Marker rescueof rpoD(Ma1)

Plasmid SD10 SD15 SD18 SD20 JH65 DA850

Approx. endpoint (aa) 255 335 410 450 565

rpoD820 + (4/3@ + (9/121) +(21/951) - w34.a - (O/370) - ww

rpoD821 + (101/273) + (7/279) n.d. + (45/251) + (Z/262) (O/514)

Recombinants between phasmid-encoded rpoD(Ma1) mutations and rpoD deletion plaamids were prepared a8 shown in Fig. 3. Endpoints of the deletions approximated from the restriction maps presented by Hu & Gross (1983) are expressed as amino acid (aa) residues from the N terminus of u’O, Regeneration of the wt rpoD allele w&9 scored by the appearance of white colonies on MacConkey maltose agar containing kanamycin and ampicillin. Numbers in parentheses show white colonies/total colonies. White colonies appear in recombinant8 with the negative control, pDAS50, at between 0 and 0.5 O/o. Plasmids were scored as rescuing a mutation ( + ) if the fraction of rpoD+ recombinant8 is at least 3 times the fraction in control infections done in parallel in at least 1 experiment. For example, the background for rpoD821 in this experiment wa* <2x 10e3, so only plasmids that gave rise to 26 x lo-” wt recombinants/Amp’ Kan’ lysogen were scored as giving marker re8cue. n.d., not determined.

J. C. Hu and C. A. Groes

22 Region 1

Region 2 homology

homology

Region homology

Region 4

3

homology

nn

-l-l

rpoD820 rpoD838 rpoDtXV

n

proposed

HTH

AV415 AV438 TWO

rpoD823

RC584

rpoDR24

RHSM

Figure 4. rpoD (Ma]) mutations lie in 2 clusters in rpoD. A representation of the rpoD gene, with the positions of rpoD(Ma1) mutations indicated as boxes. The key shows sequence changes for each allele ae the l-letter code for the wt amino acid, the mutant amino acid and the position in the a” polypeptide. For example, AV415 indicates that rpoD820 is an Ala to Val change at position 415. Regions homologous to other a factors are indicated using the nomenclature of Gribskov & Burgem (1986) by brackets over thickened portions of the sequence. Hatching indicates the 14 amino acid residues that are identical between a” and a3’.

be referred to as AV415. We chose the rpoD823 allele as the representative of the RC584 mutation. Although different isolates of the Arg to Cys change at position 584 (RC584) differ slightly in phenotype, most of the isolates are viable in the presence of wt rpoD at all temperatures, while the rpoD823 mutation is cold-sensitive under these conditions. Reconstruction experiments show that the single RC584 mutation is both necessary and sufficient for

the mutant phenotype (J. Rich, unpublished results). We believe that other isolates of RC584 may have accumulated secondary mutations that suppress the deleterious effect of the single mutation in rpoD823. We did not identify DNA sequence changes for eight mutants, all of which map to the right

of amino

acid 335. In these cases

we were unable to map the mutations precisely. These isolates were not studied further.

Table 4 Summary of rpoD(Ma1) phenotypes

Change

? P AV415 AV438 T1440 z 2 1 G5577 RC584 RC584 RH584

Isolates rpoD834,836 rpoD840,841 rpoD820 rpoD838 ~~0835 rpoD837 rpoD839 rpoD842 rpoD821 rpoD823 rpoD822,825, 826, 829-833 rpoD824, 827, 828

Maps to right of aa residue

30°C

Mal+ at 37°C 42°C

rpoD800

Complements rpoD40

335 335 410 410 410 410 410 410 565 565 565

+* +* +* +* +* +* +* +* +

+ + + k + + +

+ +*

+ + + + + + + + +

n.d. +

+ +

+* +*

inv. inv.

+ + + + + + + + inv. inv.

565

+

+

+*

inv.

inv

+

Other muc.

muc. muc. muc. ts (43°C) CS

Mutations are arranged by approximate map location in rpoD. The nature and location of amino acid (aa) substitutions is aa for Fig. 4. Map positions are for the rightmost deletion endpoint that rescued mutations, as in Table 3. + and - for ma2 expression indicate phenotypes on MacConkey maltose plates at the indicated temperatures; + * indicates the temperature at which each isolate was selected. For complementation, + indicates growth at the restrictive temperature (42”C), while inv. indicates that this combination was unable to form colonies at any temperature. muc. indicates mucoidy. ts, temperature-sensitive; cs, cold-sensitive; n.d., not determined.

rpoD Mutations

that Alter Gene Expression

dnaC’s

23

?PoD+

1 dnoG

TnlO

I. pJH62

‘*

Transduce SdeciTet’ Screen

into

1’ dnoG

Is rpoD

+ indicator

strain

and dnoG+ for rpoD

(Mal)

mutant

phenotype

Figure 5. Transfer to the chromosome. Mutations that are viable in the absence of a wild-type copy of rpd can be transferred to the E. coli chromosome. Integration of the phasmid by homologous recombination generates a duplication at the chromosomal rpoD locus. When the recombinational event. occurs to the right of the mutation, as drawn in the Figure both the wild-type dnaG allele and the mutant rpoD allele are on the same side of the I sequences as the TnlO. This recombinational event can be detected by growing phage Pl on the phasmid lysogen and transducing a AR Tet” rpoD+ recipient to Tet’ (encoded by the linked TnZO), dnaG (encoded by the integrating phasmid) and screening for the rpoD(Ma1)phenotype.

(Q Transferring the rpoD (Mal) mutations to the chromosome The presence of a wt chromosomal copy of rpoD complicates characterization of mutant phenotypes using the phasmid system, since measurements of expression from specific promoters will be the aggregate of the initiation frequencies of the two forms of Eo”. For the viable alleles, we eliminated rpoD + by crossing the mutations into the chromosome. The scheme we used is shown in Figure 5. In some fraction of phasmid-containing cells, the phasmid will integrate into the rpoD region of the chromosome, generating a duplication of the region with two copies of rpoD separated by integrated 1 DNA (Fig. 5), and linking the phasmid-encoded allele to a nearby TnlU insertion. This integration event cannot be detected in the cells where it occurs. However, the rpoD(Ma1) mutation can be cotransduced by phage Pl with flanking markers, TnlO and dnaG. Tet’, DnaG+ transductants were screened for the presence of the rpoD(Ma1) mutation. Transductants with the rpoD(Ma1) mutation

were then tested

for the ability

to transfer

the mutant phenotype by cotransduction with TnlO. Transfer of the entire cointegrate occurs infrequently due to the size of the integrated 1

sequencesrelative to the packaging limits of phage Pl. Thus, the most frequent class of recombinants will be haploid for the rpoD region. Transfer of the entire phasmid generates Kan’ transductants; these were discarded. Direct transfer of the phasmid was prevented by using a A-resistant recipient. (g) Effects of the rpoD (Mal) mutations on ma1 operon expression The rpoD(Ma1) mutations were selected to allow a malTp7 strain to grow on maltose in the absence of CAP-CAMP. To measure the effects of the rpoD(Ma1) mutations on expression of individual ma1operons, we measured j?-galactosidase synthesis in strains carrying 1acZ fusions to the m&K, malE and mulP operons. These measurements were done in the absence of maltose to avoid differences due to differential accumulation of inducer. The rpoD(Ma1) mutations have different effects on the malK, m&E and m&P operons (Table5). Mutations AV415 and T1440 increase expression of all three, but AV415 affects expression of the two operons involved in maltose transport more strongly than the malP operon, which encodes genes required for maltose metabolism. This difference is even more striking for GS577, which

24

J. C. Hu and C. A. Gross

Table 5 Expression Iininduced

A. Viable rpoD + AV415 T1440 GS577

msl operon

of ma1 operons

expression m&K:

:lacZ

malE:

:lacZ

m&P:

:lacZ

wuzlT: :lacZ

1.0 ll.l+ so+ 9.6+

(1.6) 2.4 0.5 0.9

1.0 9.0* 3.0* 11.0+

(17) 1.3 0.7 1.4

1.0 (4.5) 38+ 1.6 2.2* 1.0 1.6+ 0.4

1.0 0.7f 15* 1.45

(23) 0.1 0.3 0.1

1.0 2.5f 2.6+

(0.2) 0.7 1.1

1.0 1.5* 3.1&

(10) 0.3 0.6

1.0 0.6+ 1.4*

1.0 0.4* O.Sk

(17) 0.1 0.1

mutants

B. Non-viable h-poD+/rpoD+ 1RC584/rpoD+ 1RH584/rpoD+

mutants (1.1) 0.2 0.6

/I-Galactosidase activity was measured as a differential rate in M9 minimal medium containing glucose at 37°C (see Materials and Methods). Values for rpoD+ and IrpoD+ are arbitrarily set to 1.0, and relative expression of b-galactosidaee fusions in the rpoD mutants, determined as an average value from several experiments, are expressed normalized to the isogenic rpoD+ strain. Experimental error is expressed as the standard deviation of the normalized values. fi-Galactosidase activity, expressed in units/cell O.D. (Miller, 1972), is shown in parentheses for the rpoD+ and IrpoD’ strains.

dramatically increases expression of the malK and malE operons, but affects the malPQ operon only slightly, if at all. Since the mutations were selected for growth on maltose, the effects of the two non-viable mutations, RC584 and RH584, on uninduced ma2 expression are surprisingly small. The increases in expression of the m&E and m&K operons in these mutants are probably amplified in the presence of maltose by feedback through changes in intracellular inducer levels. Changes in rpoD could indirectly cause differences in in-vivo expression of the ma1 operons by changing expression of malT, which encodes the activator for the mu1 regulon. We tested this possibility by measuring j?-galactosidase synthesis in rpoD(Ma1) strains carrying a mdTp7-1acZ protein fusion. None of the mutants dramatically increased m&T expression (Table 5, column 4). However, the slight decrease in m&T expression with RC584 and RH584 could account for the similar decreases in malP expression by cells harboring these alleles. In the course of these measurements, we noticed that the amount of fl-galactosidase synthesis from the m&P and m&K operons in the rpoD’ controls was substantially lower in the strains containing the phasmid. We do not understand the nature of this effect. (h) Effects of the rpoD(Ma1) mutations ara and lac expression

on

that We previously observed rpoD(Alt) mutations, which were selected to increase expression of the ara operon, decreased expression of the ma1 operons (Hu & Gross, 1985). We introduced the rpoD(Ma1) mutations into strains containing an araBA: :lacZ fusion to measure their effects on transcription from ParoBAr, (Table 6A). The upstream rpoD(Ma1) mutations AV415 and TI446 do not significantly affect araBAD expression. The

downstream GS577 mutation, however, decreases araBAD expression two- to threefold. The two non-viable mutations, RC584 and RH584, were defective in arabinose metabolism on indicator plates in an araBAD+ background. However, we were unable to obtain consistent measurements using the araBA: :lacZ fusion with these rpoD(Ma1) alleles; this problem might be due to the presence of bacteriophage Mu functions. We tested whether the rpoD(Ma1) mutations affected lac expression in a strain lacking CAMP

Table 6 Effects of the rpoD(Ma1) mutations la& expression A. araBA:

and

:lacZ expression

Chromosomal rpOD+ AV415 T1440 GS577

alleles

B. lac operon Chromosomal rpoD+ AV415 T1440 GS577

expression alleles

Phasmid rpoD + RC584 RH584

on araBAD

1.0 1.0 f 1.3 f 0.4 &-

(9) 0.2 0.2 0.2

1.0 0.40+ 0.97* 0.32+

(88) 0.05 0.07 0.02

1.0 0.28f. 0.23*

(115) 0.02 0

alleles

A. p-Galactosidase activity was measured rate in M9 minimal medium containing arabinose at 37°C. Values for expression expressed as in Table 5. B. /I-Galactosidaae activity was measured rate in M9 minimal medium containing 0.2% isopropyl-thio-fl-n-gaiactoside at 37°C. Values 1acZ are expressed as in Table 5.

as a steady-state glucose and 2% of araBAD are as a steady-state glucose and 1 mMfor expression of

rpoD Mutations

that Alter Gene Expression

(Acya). Table 6B shows that four rpoD(Ma1) mutations decreased 1a.c expression 2.5 to fourfold. Because CAP-CAMP is the only transcriptional activator known to be involved in lac expression; this result suggests that these four rpoD(Ma1) mutations directly affect promoter recognition in vivo.

4. Discussion (a) Isolation of rpoD (Mal) mutations Mutations in rpoD that increase gene expression from weak promoters should allow us to identify regions of the a” polypeptide that are involved in transcription initiation. One such class of mutations, the rpoD(Alt) or alt-mutations, has been described (Silverstone et al., 1972; Hu & Gross, 1985). Our observation, that rpoD(Alt) mutations decrease expression of ma1operons, led us to predict that selection for the reciprocal phenotype, increased CAP-CAMP independent ma1 expression, might lead to the isolation of a new class of rpoD mutations. Mutations in rpoD that alter promoter selectivity are likely to have deleterious effects on cell growth. In order to obtain mutations that might not support normal transcription, we have isolated rpoD mutations on a multicopy vector in the presence of a normal chromosomal copy of the gene. This approach requires that the desired phenotype be codominant or dominant. By constructing a clone of the cr operon as a hybrid between the plasmid ColEl and phage ,I we were also able to exploit advantages of both phage and plasmid systems. The phasmid A pJH62 can be grown lytically by methods normally used for propagation of 1, or can be maintained as a lysogen in a plasmid state. Since replication of the plasmid origin is not controlled by 1 immunity, lysogens can be formed efficiently in homoimmune (imm21) cells and selected by resistance to kanamycin. Packaging of the phasmid allows it to enter I-sensitive cells efficiently. Phasmid DNA can be amplified by chloramphenicol treatment of lysogens. A similar plasmid-phage hybrid has been described (Elledge & Walker, 1985). Localizing mutagenesis to the plasmid IZpJH62 increases the frequency of rpoD mutations between ten and lOOO-fold. Using the phasmid system described above, we isolated 23 independent mutations in rpoD that allowed a Acya malTp7 strain to grow on minimal maltose plates. Several of these isolates failed to complement the rpoD800 mutation: in fact, they were incompatible with this allele even under conditions normally permissive for rpoD800. One mutant was cold-sensitive even in the presence of a chromosomal copy of rpoD+ . (b) Mapping mutations on the phasmid vector Mutations on the phasmid cannot be mapped directly by marker rescue with rpoD deletion

25

plasmids. Recombination between phasmids and the chromosomal rpoD will also rescue the mutations. Since homology between the phasmid and the chromosome is large, and the homology between deletion plasmids with endpoints near the mutation will be small, recombination with the chromosome could be up to lOOO-fold more frequent than recombination with the deletion plasmids. We developed a mapping technique that enriches for recombinants between the phasmid and our mapping plasmids by selecting for formation of a cointegrate. This enrichment for the recombinant class allows us to detect marker rescue events with deletion endpoints very near a mutation. Deletion endpoints within 50 base-pairs generate wt cointegrates as 0.1 to 1y. of the colonies scored. Without this enrichment, wt recombinants would be expected at 10e6 or lo- ’ (Hu & Gross, 1983). This technique is especially useful for the study of essential genes, where removing homologous chromosomal DNA is impractical. We anticipate broader application of this approach, as it makes it feasible to closely map mutations in cases where the mutant and wt phenotypes can be detected only by screening individual colonies. Since deletion endpoints within less than 1 kb from our mutations generate wt cointegrates as 25 to 3Oyc of the colonies, mapping at this resolution could even be done by immunological or biochemical techniques that require preparation of crude lysates.

(c) Structural changesin the mutants Mapping against deletions in rpoD divided the rpoD(Ma1) mutations into two groups, a cluster near amino acid residues 400 to 450, and a cluster downstream from residue 560. DNA sequencing identified six different mutational changes that lead to the Mal+ phenotype. Amino acid sequence comparisons among o factors have identified regions of the ISpolypeptides that are more or less conserved (Landick et al., 1984; Stragier et al., 1985; Gribskov & Burgess, 1986). Both clusters of rpoD(Ma1) mutations fall in regions conserved among cr factors. Amino acids sharing homology with residues 384 to 456 of 0” have been found in seven other 0 factors (Gribskov & Burgess, 1986; Binnie et al., 1986). One mutation in the upstream rpoD(Ma1) cluster, AV415, falls in the most highly conserved part of this region, while the other upstream rpoD(Ma1) mutations, TT440 and AV438, lie nearby in a somewhat less-conserved amino acid sequence. The rpoD(Ma1) mutations of the downstream cluster, GS577, RC584 and RH584, lie in a conserved region of o that resembles the a-helixreverse turn-a-helix (or HTH) DNA binding motif found in several bacterial DNA hinding proteins. The concordance between the amino acid sequences identified by homology to DNA binding proteins and residues identified by the mutations suggests

26

J. C. Hu and C. A. Gross

this region of a” may, indeed, be involved in direct contacts with promoter DNA.

that

(d) Ejfects of rpoD(Ma1) mutations on ma1 expression Comparing the effects of the rpoD(Ma1) mutations on the malK, malE and malP operons leads us to draw the following conclusions. (1) Some of the mutant Q factors affect transcription of one or more of the ma1 regulon promoters independently of effects on expression of m&T itself. AV415 increases expression of all three w&T-dependent operons without increasing malT synthesis. RC584 and RH584 increase transcription of malK or malE despite decreasing n&T synthesis. (2) Expression of the three m&T-dependent promoters is not affected co-ordinately by some of the rpoD(Ma1) mutations. Comparing the effects of the T1440 and GS577 mutations suggests that either the former is selectively inhibited or the latter is selectively stimulated in activation of the malK and malE promoters. (3) None of the mutations increases malT expression dramatically; however, we do not know how large a change in m&T expression would be required to account for the Ma1 phenotypes of the mutants. Although the RC584 and RH584 mutations confer a Mal+ phenotype on malTp7 strains, their effects on ma1 expression are small. This suggests that strains carrying malTp7 are very close to being able to utilize maltose in the absence of CAPCAMP. In the presence of maltose, a small intrinsic difference in malK expression caused by RC584 and RH584 could be amplified by feedback due to transport of the inducer (Novick & Weiner, 1957). Alternatively, the Mal+ phenotype could be largely due to indirect effects of the mutants on maltose transport, perhaps by affecting expression of other membrane-associated functions. In this case, although the small increase in ma1expression would still be a real effect of the rpoD(Ma1) mutants, a comparable increase in ma1 expression in rpoD+ cells would in itself not be sufficient for a Mal+ phenotype. The Mal+ phenotype in rpoD(Ma1) mutants requires elevated MalT; the mutations do not allow malTp+ cells to grow on maltose. This indicates that ma1 transcription in the mutant cells is activated by MalT, and initiates at the same sites in mutant and rpoDf cells. Mutations that affect CAP-independent, malT-dependent initiation could thus affect either interaction of RNA polymerase with promoter DNA or with MalT. (e) Pleiotropic effects In addition to their effects on ma1expression, the rpoD(Ma1) mutations have pleiotropic effects. Some of the mutations in the upstream cluster cause mucoidy. The mutations in the downstream cluster decreasearaBAD expression.

The two non-viable mutations, RC584 and RH584, prevent growth in rpoD800 cells even under permissive for rpoD800. conditions normally Presumably, these mutations either prevent expression of essential functions or overexpress some genes in a manner that becomes toxic. We favor the former possibility, since the presence of a wt copy of rpoD on the chromosome relieves the growth inhibition by RC584 and RH584, above 37°C. Four rpoD(Ma1) mutations decrease expression from PIoc (Table 6). The magnitude of the decrease in lac expression is striking for RC584 and RH584, since the effects of these mutations must be measured in the presence of wt a” synthesized from the chromosome. Based on our estimates of relative gene expression, chromosomally encoded 0” could account for 15 to 20% of total transcription; thus utilization of the lac promoter by RC584 and RH584 could be lower than the apparent three to fourfold decrease below rpoD+. The decreases in lac expression seen in a strain lacking CAMP (Acya) are likely to reflect a change in how the mutant RNA polymerase interacts with promoter DNA, since no factor other than CAPCAMP is known to be required for lac transcription. If the rpoD(Ma1) mutations affect RNA polymerasepromoter DNA interaction, this should be observable on transcription from other promoters. Such a generalized effect may underlie the coldsensitive, dominant lethality of RC584. Experiments to determine the sequence specificity of the mutant Q factors on the P22 ant promoter and the lac promoter will be described elsewhere (D. Siegele et al., unpublished results). It is unclear whether the Ma1 phenotype of the mutants will be explained solely by effects on DNA contacts. Our finding that the rpoD(Ma1) mutations affect factor-independent transcription from the lac promoter is not mutually exclusive with the possibility that direct protein contacts between RNA polymerase and MalT are altered at ma1 promoters. Certainly, if the effect of the mutations is mediated by conformational changes in holoenzyme, these could affect contacts with both promoter DNA and activators. We can also imagine some amino acid side-chains making bidentate contacts involving both the activator and DNA, especially if the activator and RNA polymerase are bound to the same sequences on opposite faces of the DNA helix. Evidence for this type of arrangement has been presented for other activators (Ho et al., 1983; Shanblatt 6 Revzin, 1986), and the proximity of putative MalT binding sites to the -35 sequences of the ma1 promoters suggests a similar topology. We thank Qian XiaoHong and Willy Walter for their invaluable help with assays, and Dave Straus for the Western blot experiments. We thank Robert Glass, Richard Losick and members of the Gross laboratory for their comments on the manuscript. This research was supported by the College of Agriculture and Life Sciences of the University of Wisconsin-Madison and by National

rpoD Mutations Institutes of Health grant AI19635 supported by a Research Career from NIH.

that Alter Gene Expression

(C.A.G). C.A.G. was Development award

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27

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Edited by M. Gottesman