Region 1 of σ70 is required for efficient isomerization and initiation of transcription by Escherichia coli RNA polymerase 1

J. Mol. Biol. (1997) 267, 60±74

Region 1 of s 70 is Required for Efficient Isomerization and Initiation of Transcription by Escherichia coli RNA Polymerase Christina Wilson and Alicia J. Dombroski* Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, 6431 Fannin Houston, TX 77030, USA

The sigma (s) subunit of prokaryotic RNA polymerase is essential for promoter recognition. s Factor directs the RNA polymerase core subunits (a2bb0 ) to the promoter consensus elements and thereby confers selectivity for transcription initiation. The N-terminal domain (region 1.1) of Escherichia coli s70 has been shown to inhibit DNA binding by the Cterminal DNA recognition domains. Since DNA recognition is the ®rst step of transcription initiation, we predicted that the N-terminal domain of s70 may also in¯uence the initiation of transcription by holoenzyme (a2bb0 s). N-terminally truncated s70 derivatives were used to reconstitute holoenzyme, and transcription by the mutant holoenzymes was analyzed in vitro. Deletion of the ®rst 75 to 100 amino acids of s70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA- strand-separated open complex, as well as a reduced ef®ciency of transition from the open complex to a ternary initiated complex. These effects were partially reversed by the addition of a polypeptide containing region 1.1 in trans. Therefore, region 1.1 not only modulates DNA binding but is important for ef®cient transcription initiation, once a closed complex has formed. A deletion of the ®rst 133 amino acids, which removes regions 1.1 and 1.2, resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation. # 1997 Academic Press Limited

*Corresponding author

70

Keywords: s ; region 1; transcription initiation; isomerization

Introduction The sigma (s) subunit imparts the ability to recognize promoter DNA sequences to the core subunits (a2bb0 ) of prokaryotic RNA polymerase, and thus is responsible for the speci®city of transcription initiation (Travers & Burgess, 1969; Losick & Pero, 1981). s Factors are grouped into two families based on homology to either s70, the major vegetative s factor of Escherichia coli, or to s54, the s factor involved in transcription of genes in the nitrogen regulon (reviewed by Merrick, 1993). The s70 family, the larger of the two s families, has been subdivided into the functionally distinct primary and alternative s factors (Stragier et al., 1985; Helmann & Chamberlin, 1988). Primary s factors are indispensable for the maintenance of basal gene expression in vegetative cells, while alternative s factors recognize specialized subsets of proAbbreviations used: FL, full-length; NTP, nucleotide triphosphate. 0022±2836/97/110060±15 $25.00/0/mb970875

moter sequences. This extensive variety of s factors provides the bacterial cell with a powerful means of directing gene expression in response to speci®c environmental conditions. The speci®city of promoter recognition for the different s factors is determined by two separate DNA recognition domains, which contact speci®c base-pairs in the consensus promoter hexamers (reviewed by Gross et al., 1992, Figure 1a). In both the primary and the alternative s factors, region 4.2 recognizes the ÿ35 consensus sequence and region 2.4 recognizes the ÿ10 consensus sequence (Gardella et al., 1989; Siegele et al., 1989; Zuber et al., 1989; Daniels et al., 1990; Waldburger et al., 1990; Dombroski et al., 1992). Thus, the s subunit is responsible for interacting directly with the promoter DNA and aligning RNA polymerase in preparation for transcription initiation. The s subunit also appears to be required for transcription initiation events beyond DNA recognition, speci®cally for open complex formation. # 1997 Academic Press Limited

61

70 Region 1 and Transcription Initiation

Figure 1. Structure of s70 and derivatives. a, Linear diagram of s70. Regions 1 through 4 denote highly conserved blocks of amino acid sequence (Lonetto et al. 1992). Subregions are shown by divisions within the main regions. Regions 2 and 4 are involved in recognition of the ÿ10 and ÿ35 promoter DNA consensus elements. A stretch of residues involved in core binding extends into region 2. Region 1.1 is present only in the primary s factors. b, s70 derivatives. Linear diagrams show amino acid residues remaining in s70 N terminal truncation derivatives. Each s factor derivative is fused at the N-terminus to the following amino acid sequence: MRGSHHHHHHGSSGLVPRGSGLGTRL. The region 1.1 polypeptide has the following N-terminal fusion sequence: MGSSHHHHHHSSGLVPRGSHMLE. The sixconsecutive histidine residues are used for af®nity puri®cation (see Materials and Methods).

Prokaryotic transcription initiation is a multistep process that begins with the binding of holoenzyme (a2bb0 s) to the promoter, where it establishes contacts with template DNA upstream of the transcription start site in an initial closed complex designated RPc1. RPc1 isomerizes to a second closed complex, RPc2, in which RNA polymerase interaction with the template extends to contacts downstream of the transcription start site (Record et al., 1996). Promoter DNA is heteroduplex in nature in both RPc1 and RPc2. DNA strand opening accompanies formation of the ®rst open complex (RPo1), which isomerizes to a second open complex (RPo2). In the presence of initiating nucleotide triphosphates (NTPs), RPo2 complexes progress to RPinit complexes, which are stable and competent to begin processive RNA synthesis. The initial transcribing complex synthesizes abortive RNA transcripts of two to 12 nucleotides in length, followed by s factor release and entry into the elongation mode of RNA synthesis. The lPR promoter was used to elucidate many details of this pathway (reviewed by Record et al., 1996). The core RNA polymerase subunits cannot catalyze strand opening on covalently closed circular DNA in the absence of s70 (Saucier & Wang, 1972).

More recent studies of an alternative s factor, sE (Jones & Moran, 1992; Jones et al., 1992) and the primary s factor, sA (Juang & Helmann, 1994; Rong & Helmann, 1994) from Bacillus subtilis, implicate the s subunit in strand opening by demonstrating that mutations in region 2.3 of s (Figure 1 a) can affect the DNA melting stage of initiation. The mechanism of s involvement in this process, however, is not yet clear. The structural differences between primary and alternative s factors, as well as differences in promoter con®guration (DeHaseth & Helmann, 1995), suggest that the mechanism of open complex formation may vary for different s factors. Here, we limit our analysis to the primary E.coli s factor, s70. The N-terminal domain of s70 can be divided into two subregions, 1.1 and 1.2, which span residues 1 through 100 and 101 through 133, respectively (Figure 1a). Until recently, region 1.1, which is present only in the primary s factors, had not been assigned a function (Gribskov & Burgess, 1986; Lonetto et al., 1992; Helmann & Chamberlin, 1988). Dombroski et al. (1992, 1993) demonstrated that region 1.1 is involved in modulating the DNA binding activity of free s70. Since the DNA binding function of s is directly responsible for the promoter recognition step of transcription initiation, and since region 1.1 in¯uences this activity, we hypothesized that region 1.1 might also play a role in the initiation process. Region 1.2, while highly conserved among almost all s factors, has not yet been assigned a function (Lonetto et al., 1992). Here, we explore the functional role of regions 1.1 and 1.2 of s70 in transcription initiation. We present evidence that amino acids between 50 and 100 of region 1.1 are required both for ef®cient transition from the closed to the open promoter complexes and for the formation of the initial transcribing complex. Additionally, we show that open complexes cannot form in the absence of region 1.2.

Results Conserved region 1.1 of s 70 plays a role in transcription initiation In order to address whether the N terminus plays a role in the process of transcription initiation, several s70 derivatives were constructed. Our analysis included a series of truncations which remove 50, 75, 100, and 133 amino acids, respectively from the N terminus of s70 (Figure 1 b).
100 lacks region 1.1 entirely, and
133 lacks both regions 1.1 and 1.2. DNA fragments of appropriate lengths from the rpoD gene (encoding s70) were inserted into expression vectors, as described in Materials and Methods. The synthesis of full-length (FL) and truncated s factors was induced with isopropyl thio-b-D.-galactoside (IPTG). Since these polypeptides fractionated as inclusion bodies with the membrane pellet, they were puri®ed under denaturing conditions, using a batch method and a

62

Figure 2. In vitro run-off transcription assays . Reconstituted holoenzymes containing truncated s70 derivatives were used to transcribe linear templates containing the Ptac and lPR promoters. The 100 and 80 nucleotide Ptac and lPR transcripts are shown on an 8% denaturing polyacrylamide gel. The bar plots (Ptac, hatched; lPR, ®lled) show the fraction of transcripts synthesized by the mutants as compared to the FL holoenzyme under the same conditions. Values were averaged from at least three separate experiments and then normalized. Error was less than 12% (shaded).

Ni2 ‡ af®nity resin, and refolded after elution. The region 1.1 polypeptide was puri®ed as a soluble protein under non-denaturing conditions, following the same protocol with the omission of urea from the buffers. The puri®ed s derivatives were used to reconstitute holoenzyme for functional characterization. We ®rst assessed whether N-terminally truncated s70 derivatives could enter into the processive, elongation phase of transcription by measuring the synthesis of an 80-nucleotide long run-off transcript from the lPR promoter (Figure 2). lPR has been thoroughly characterized in vitro (Bernard & Meares, 1986; Hawley & McClure, 1980; Roe et al., 1984; Roe & Record 1985; Suh et al., 1992, 1993; Craig et al., 1995) and was therefore selected for our transcriptional analysis. We will continue to refer to holoenzymes carrying the truncated s factors simply as FL,
50,
75,
100, and
133.
75 and
100 exhibited a 75% and 90% reduction, respectively, in the formation of run-off transcripts, while
50 functioned as well as, or better than FL in the same assays. The largest truncation,
133 did not show activity above background (data not shown). Transcription was also tested on a second promoter, Ptac, in order to determine whether the observed behavior was promoter speci®c (Figure 2). The activities of the truncated s derivatives on Ptac were not signi®cantly different from those on

70 Region 1 and Transcription Initiation

lPR. All further analysis was performed using lPR. Transcription by FL and
100 was also examined in single-round assays, to eliminate the possibility that the truncations might be impaired for s recycling. With this limitation, FL and
100 maintained the same relative activity as seen in multiple round transcription assays (data not shown). These experiments indicated that the reduction in transcription for
75 and
100 was not occurring at the stage of s release and that it was probably occurring at an earlier step in initiation. Support for the idea that the truncated s factors are de®cient in initiation prior to promoter clearance was provided by examining the behavior of FL and each s70 derivative in abortive transcription. Reaction conditions established for run-off transcription were maintained in this analysis.
75 and
100 exhibited an 80 to 90% reduction in the synthesis of a three-nucleotide abortive product as compared to the FL protein (Figure 3). The relative amounts of the abortive transcripts were similar to those measured in run-off transcription assays. The total distribution of abortive RNA products synthesized by holoenzyme formed with truncated s derivatives, relative to the run-off transcript, was determined using quantitative analysis of abortive transcription (Hsu, 1996). This assay allows the examination of run-off and abortive products in the same experiment, and provides an indication of the ef®ciency of promoter clearance. We repeated our analysis of the N-terminal truncations using this method and con®rmed that the deletions

Figure 3. In vitro abortive transcription assays. Reconstituted holoenzymes containing truncated s70 derivatives were assayed for the ability to synthesize a 3-nt abortive transcript from linear template containing lPR. The total transcript synthesized by holoenzymes containing each s70 truncation was visualized on a 24% denaturing polyacrylamide gel. The bar plots show the fraction of total 3-nt product as compared to transcript formed by FL. Values were averaged from at least three separate experiments and then normalized. Error was less than 15% (shaded).

63

70 Region 1 and Transcription Initiation Table 1. Kinetic constants for abortive initiation at lPR by N-terminally truncated s factors s70 Derivative

KB  10ÿ8 (Mÿ1)

kf  102 (sÿ1)

KBkf  10ÿ7 (Mÿ1sÿ1)

2.8 2.3 3.8 3.3 5.2

4.0 4.5 0.2 0.2 0.6

1.09 1.03 0.09 0.08 0.31

FL
50
75
100
100 ‡ reg 1.1

Values were calculated using tau plot analysis (McClure 1980), which assumes a simple two-step mechanism: k1

kf

R ‡ P „ RPc ! RPo: kÿ1

impede both run-off and abortive RNA synthesis to the same extent, but do not affect promoter clearance. In addition, the size distribution of abortive transcripts was similar for FL and each truncation (data not shown). Taken together, the results of run-off and abortive transcription analysis imply that residues in the Nterminal domain of s70 are required at a stage of transcription initiation which precedes promoter clearance. The most dramatic effect occurred when amino acids between 50 and 75 were removed, suggesting functional signi®cance for that region.

N-terminal truncations affect the process of isomerization during initiation To determine more precisely how the N terminus of s70 affects early steps in initiation, a kinetic analysis of abortive initiation was performed in vitro. Using tau plot analysis (McClure, 1980), we measured the initial binding constant, KB (k1/ kÿ1), and the isomerization rate constant, kf for holoenzyme formed with FL and the truncated s derivatives,
50,
75, and
100 (Table 1). The rate constant, kf, represents all of the intermediate complexes from RPc1 through RPinit, since the tau plot analysis assumes a simple two-step mechanism (see Table 1). Under ®rst-order reaction conditions (RNA polymerase in excess), transcription reaches a steady state after a lag time (tau). Tau represents the time required for polymerase to bind, isomerize, and begin transcription at a promoter. A plot of tau as a function of 1/[RNA polymerase] allows the determination of KB and kf. The slope of the tau plot represents the inverse of promoter strength (KBkf), while the y-intercept is the inverse of the forward rate constant (kf). The KB values for FL,
50,
75, and
100 were not signi®cantly different, implying that initial binding to the promoter is similar for holoenzyme formed with each s factor derivative. The kf of
50 was also equivalent to that of FL.
75 and
100, in contrast, showed a marked reduction in kf by a factor of 20 (Table 1). The overall ef®ciency of promoter-utilization, re¯ected by KBkf, is thereby reduced by factors of 11 and 13, for
75 and
100, respectively. These results argue that amino acids between 50 and 75 are

essential for ef®cient open complex formation, but are not required for closed complex formation. To better understand the isomerization defect of the deletion mutants, and to identify intermediate steps affected by the removal of region 1.1 or region 1.2, we analyzed the DNase I cleavage patterns of complexes formed between holoenzymes containing FL and two of the truncated s70 derivatives (
100 and
133), with lPR template DNA. The FL,
100 and
133 holoenzymes were chosen for this analysis, as representative of the three classes of behavior observed in transcription (normal, reduced, and no transcription). s70 Holoenzyme forms complexes with the lPR promoter which extend from ÿ55 to ÿ5 in RPc1, and ÿ55 to ‡20 in RPc2. The open complexes (RPo1, RPo2, and RPinit) also show extended protection from ÿ55 to ‡20 (Craig et al., 1995), reviewed by Record et al., 1996). Our analysis does not discriminate between RPo1 and RPo2, so we refer to these complexes collectively as RPo. Since tau plot analysis indicated that progression from RPc1 to RPinit is slow for the truncation derivatives, we evaluated DNase I footprints at two and 40 minutes following addition of RNA polymerase to DNA. After two minutes, the FL holoenzyme protected the non-template strand of the DNA in a region extending from ÿ55 to ‡20, with enhancements at positions ÿ43 and ÿ45, typical of RPc2 or RPo (Craig et al., 1995; Record et al., 1996). Holoenzyme containing
100 and
133 protected the promoter DNA from ÿ55 to ÿ5 (Figure 4a), as observed for RPc1 (Record et al., 1996), with a slight enhancement seen for the
133 complex, at position ‡3.
100 showed some very weak protection in the ÿ5 to ‡20 region of the promoter. We conclude that
100 and
133 are forming predominantly the ®rst closed complex (RPc1) at two minutes. After 40 minutes,
133 complexes remain in RPc1, with additional enhancement seen at position ÿ12, while
100 complexes now show the extended footprint, with protected regions between ÿ55 and ‡20 (Figure 4b), characteristic of RPc2 or RPo (Craig et al., 1995; Record et al., 1996). This 40 minute
100-DNA complex extends to ‡21, slightly farther than the FL complex (Figure 4b). Neither core RNA polymerase alone nor s factor alone formed a DNase I footprint (Figure 4c). Open complexes are known to be stable to challenge with the polyanionic competitor, heparin, while closed complexes are unstable (Melancon et al., 1982; Roe et al., 1984). As expected, FL complexes were stable to heparin challenge at two and 40 minutes, indicating that FL rapidly forms open complexes (Figure 4a and b).
100 Complexes were sensitive to heparin challenge at two minutes but not at 40 minutes, demonstrating a slower progression from RPc1 to RPo (Figure 4a and b).
133 complexes were sensitive to heparin at both two and 40 minutes, suggesting that the formation of RPo is either extremely slow or unattainable. The DNase I experiments do not directly show whether the extended RNA polymerase-DNA

64

70 Region 1 and Transcription Initiation

Figure 4. Dnase I footprints of FL and mutant holoenzyme-lPR complexes. a. Complexes formed on the non-template strand of the lPR promoter at two minutes in the absence and presence of heparin. b, Complexes formed at 40 minutes in the absence and presence of heparin. c, Negative controls with
133,
100, and FL s factors alone (designated s
133, s
100, and sFL), and core RNA polymerase alone, with the FL holoenzyme footprint shown for reference. Positions are indicated relative to the start site (‡1) of transcription.

complexes are actually strand-separated, as in RPo, or base-paired as in RPc1 or RPc2. We therefore performed potassium permanganate (KMnO4) footprinting. KMnO4 preferentially modi®es thymine residues present in single-stranded regions of DNA and renders the modi®ed bases susceptible to cleavage by piperidine (reviewed by Record et al., 1996). Single-stranded regions are weakly detectable in
100 complexes at two minutes (11% cleaved), but by 40 minutes, the amount of singlestranded DNA (54% cleaved) actually exceeded that observed for FL complexes by 1.5-fold (Figure 5), arguing that
100 is slower than FL in progressing from RPc1 to open complexes. The positions sensitive to piperidine cleavage correlate with the positions detected by Suh et al. (1993). Single-stranded DNA detected in
133 complexes at two minutes (1% of input DNA cleaved) and 40 minutes (5% cleaved) is negligible when compared

with that detected in the FL complexes formed at the same time points (34% and 36% cleaved, respectively; Figure 5), supporting the idea that the majority of the
133 complexes are halted at RPc1. We note that the T at ÿ7 becomes susceptible to a minor amount of cleavage in the
133 complexes, but not in the FL or
100 complexes, implying that
133 forms a slightly different structure with the DNA. In summary, the results presented thus far indicate that
100 holoenzyme clearly has the potential to form open complexes with an extent of strand melting at least equivalent to open complexes formed by holoenzyme containing FL s70. If
100 is impaired solely by a slower rate of strand opening, then we predicted that a long preincubation with template, prior to addition of initiating nucleotides, should allow nearly full recovery of transcript production. However, this was not the

65

70 Region 1 and Transcription Initiation

complexes were stable. The instability of
100 complexes to high salt is due to reduced ability to initiate RNA synthesis because even if given suf®cient time to form open complexes, in the absence of heparin, production of abortive transcripts is signi®cantly impaired. It therefore appears that even though
100 polymerases eventually form open complexes, these complexes are inef®cient at progressing to stable, transcriptionally active complexes. Region 1.1 partially restores transcriptional activity of
100 in trans

Figure 5. Potassium permanganate (KMnO4) footprints of FL and mutant holoenzyme-lPR complexes. Complexes were formed for two and 40 minutes. Positions of piperidine sensitivity are indicated (‡2, ÿ3, ÿ4, ÿ7, and ÿ10). The percentage of cleaved DNA relative to total DNA recovered is shown in the bottom panel. Bands were quanti®ed directly using a Packard Instantimager.

case. Even after 40 minutes, where the KMnO4 footprinting showed
100 polymerase to have formed open complexes similar to FL, production of transcript remained signi®cantly lower (24% of FL). Thus, we hypothesized that
100 may be additionally affected in its ability to progress from RPo to RPinit. Taylor & Burgess (1979) have shown that initiated open complexes, (now called RPinit), are stable to a high salt wash (0.8 M NaCl) during nitrocellulose ®lter binding, while complexes formed in the absence of nucleotides are unstable. Roe et al. (1984) used this method to select for active open complexes during ®lter binding. We tested the stability of the various mutant polymerases to high salt wash, following the addition of the ®rst three nucleotides. Complexes retained on the ®lter represent the percentage of RPinit complexes formed at equilibrium. 46% of the FL complexes were stable to a high salt wash, as expected (Roe et al., 1984; Record et al., 1996), while only 8% of the
100

We next asked whether the missing amino acids of region 1.1 would affect transcription when added back to the various truncated s factors. Earlier work by Dombroski et al. (1993) demonstrated that region 1.1 inhibits the DNA binding activity in vitro of polypeptides containing the C-terminal DNA binding domains of s70 when provided in trans. Following this same logic, we tested whether region 1.1 could stimulate transcription by holoenzymes formed with the N-terminally truncated s70 derivatives. First, we added puri®ed region 1.1 polypeptide, in increasing molar excess, to FL and
100, in an incubation step prior to the addition of the core subunits. Following core addition, reactions were incubated at 37 C, prior to addition of NTPs, in order to allow the formation of competent initiation complexes. Quantitation of the 80nucleotide RNA product revealed that
100 activity was enhanced, while FL transcriptional activity was inhibited, by increasing amounts of region 1.1 (Figure 6). A 60 to 80% stimulation of
100 activity was seen in the range of 150 to 200-

Figure 6. Effect of adding increasing amounts of region 1.1 in trans on run-off transcription using lPR. Region 1.1 was added in molar excesses ranging from 15 to 200 (x-axis). Under these conditions, run-off transcription by holoenzyme containing
100 was enhanced, while transcription by holoenzyme containing FL was inhibited. Percent change in transcript synthesis denotes the increase or decrease relative to the transcript synthesized in the absence of region 1.1 addition in trans.

66

Figure 7. Comparison of the effect of region 1.1 addition in trans on run-off versus. abortive transcription by s70 N-terminal truncations. Region 1.1 was added at 150fold molar excess. Percent change in transcript synthesis denotes the increase or decrease in transcript synthesis relative to transcript synthesized in the absence of added region 1.1. Values were averaged from at least three separate experiments and then normalized. Error was less than 12% for abortive and less than 16% for run-off transcription.

fold excess of region 1.1 relative to s factor. A maximum of 30% inhibition was seen for FL activity under the same conditions (Figure 6). We believe that the large excess of region 1.1 was required to approximate the localized concentration of region 1.1 present in cis in the intact protein. Though a signi®cant enhancement of
100 activity was seen in the range of 30-fold excess, we chose to conduct subsequent experiments at 150-fold excess, a concentration which expressly enhanced and inhibited the activities of
100 and FL, respectively. The impact of region 1.1 addition on the synthesis of both run-off and abortive transcripts was compared for FL and the transcriptionally active truncated s derivatives (
50,
75, and
100). The effect of region 1.1 addition was similar on both run-off and abortive transcript synthesis (Figure 7). Of the proteins examined, only
100 consistently showed a restoration of transcription ability in response to region 1.1 addition in trans. FL,
50, and
75 exhibited an inhibitory response to region 1.1, the basis of which remains unclear. These results indicate that region 1.1 can interact with
100 in trans, and that the conserved regions of the s subunit can function as separate, modular domains. To test whether the addition of region 1.1 speci®cally reverses the isomerization defect (kf) for
100 described above (Table 1), abortive initiation kinetic analysis was repeated for holoenzyme containing
100, with region 1.1 added in trans. A comparison of tau plots generated for
100 in the presence and absence of region 1.1 revealed a trend that indicates partial reversal of the original defect. This translates into a slight increase in KB and a threefold increase in kf, relative to the kinetic

70 Region 1 and Transcription Initiation

analysis in the absence of added region 1.1 (Table 1). The increase in kf, could be due to enhanced isomerization from RPc1 to RPo, or from RPo to RPinit. As judged by nitrocellulose ®lter binding assays performed under high salt conditions (see above), the addition of region 1.1 in trans to
100 holoenzyme, at 50 and 150-fold molar excess, failed to stabilize the
100 RPinit complexes. The enhancement of
100 transcription by region 1.1 therefore appears to be due to stimulation of a transition which precedes the formation of RPinit, presumably RPc1 to RPo. A comparison of the time-dependent formation of
100 open-complexes by KMnO4 footprinting analysis reveals an approximate twofold stimulation in the rate of strand melting, when region 1.1 is added in trans, reinforcing the idea that enhancement is due to stimulation of the RPc1 to RPo transition (Figure 8). We were unable to address the mechanism of inhibition of FL by region 1.1 in trans using abortive kinetic analysis due to the variability of the measurements. s factor structure and role of region 1.1 in vivo We believe that the truncated proteins are properly folded and that the observed defects are not due to large perturbations in structure, since proteolytic digestion of FL and truncated s factors in vitro with trypsin yielded similar fragment patterns (data not shown). In addition,
50,
75 and
100

Figure 8. Effect of region 1.1 addition on time-dependent KMnO4 footprint of
100 holoenzyme-lPR complexes.
100 holoenzyme-lPR complexes were formed in the absence and presence of added region 1.1. Sample aliquots were removed and analyzed for strand opening over a 25 to 30 minute time interval. a, Resolution of piperidine sensitive DNA over time (minutes). b, Quanti®cation of the fraction of DNA cleaved (in open complexes) relative to the total DNA recovered. Cleavage positions correspond to ÿ4, ÿ3, and ‡2 relative to the start site of transcription (‡1).

67

70 Region 1 and Transcription Initiation

ility and/or other aspects of s70 activity in vivo. Each histidine-tagged s70 derivative was also expressed in an E. coli strain carrying the chromosomal copy of rpoD under the control of a synthetic trp promoter. Under conditions favoring repression of the chromosomal rpoD gene, only the FL s70 derivative restored growth. The failure of
50 to complement, in spite of its ability to function in vitro, is probably therefore due to the removal of amino acids that are essential for viability.

Figure 9. Core binding competition assay. Equal amounts of FL and either
100 or
133 s factors were allowed to compete for binding with a ®xed amount of core RNA polymerase. Complexes were probed for strand opening at two minutes, when the relative activities of FL,
100 and
133 are readily distinguishable (see Materials and Methods). The insert panels show the cleavage products resolved on an 8% denaturing polyacrylamide gel. The shaded bars show the amount of the input DNA cleaved, normalized to the fraction of DNA cleaved in complexes containing FL holoenzyme alone. Values were averaged from three separate experiments. The error (white) was 9% for complexes containing FL and
100 holoenzymes and 4% for complexes containing FL and
133 holoenzymes.

exhibited similar DNA-binding activities, signifying no serious alteration in protein conformation (data not shown).
100 and
133 do not have reduced af®nity for core since they both compete with FL s70 for core binding. KMnO4 footprinting analysis of holoenzyme formed with a mixture of FL and the truncated s factors shows the expected reduction in open complex formation (Figure 9). Additionally, glycerol gradient fractionation did not reveal differences in the association of the deletion derivatives with the core subunits (data not shown), and because these truncations do not remove the de®ned core binding region of s70, we did not expect them to be de®cient in core binding (Lesley & Burgess, 1989; Figure 1 a). The histidine-tag does not interfere with s70 function in vivo, since expression of FL complemented the temperature-sensitive (ts) growth phenotype of a strain carrying a mutant s70 allele, rpoD800 (also rpoD285). None of the truncated s70 proteins, however, reversed the ts phenotype. Since each s derivative functioned equally well at 44 C and 37 C for transcription in vitro, we do not suspect that the failure to complement rpoD800 at 44 C is due to the thermolability of the truncated proteins. The
50 protein, however, is more proteolytically susceptible than FL when expressed in vivo at 44 C (data not shown). This observation could account for the inability of
50 to rescue the rpoD800 mutation at the non-permissive growth temperature. Thus, region 1.1 may be essential for protein stab-

Discussion The s subunit of prokaryotic RNA polymerase is involved in several key steps in basal transcription initiation. s Factors mediate the initial recognition of promoter DNA and have been implicated in open complex formation. Several mechanisms have been described for the pathway of open complex formation on s70 promoters, involving the localized melting of approximately 12 bp of promoter DNA (DeHaseth & Helmann, 1995; Record et al., 1996). The participation of s70 in DNA melting was ®rst proposed to explain why s factor was dispensable for initiation from templates which already contained single-stranded regions, but was required to initiate from promoters composed of intact duplex DNA (Hinkle & Chamberlin, 1970). Physical evidence for contact between s70 and the non-template strand of the melted promoter region was obtained with UV crosslinking experiments (Buckle et al., 1991; Simpson, 1979; reviewed by DeHaseth & Helmann, 1995). Sequence analysis of several s factors has revealed a cluster of conserved aromatic and basic amino acid residues in region 2 of s70, which were postulated to interact with single-stranded DNA, based on some similarity to single-stranded nucleic acid binding proteins (Helmann & Chamberlin, 1988). The hypothesis that the function of s factor in strand melting is to stabilize single-stranded DNA in the open complex, via interaction with the nontemplate strand, emerged from these observations (DeHaseth & Helmann, 1995). Precisely how s factor participates in DNA melting is not known, but several groups have implicated region 2.3 in this process. A single amino acid substitution in region 2.3 (Figure 1a) of an alternative B. subtilis s factor, sE, was shown to prevent the transition between closed and open complex (Jones & Moran, 1992; Jones et al., 1992). Rong & Helmann (1994) described several amino acid substitutions in region 2.3 of sA, the B. subtilis s70 homolog, which speci®cally hampered open complex formation in vitro. On the other hand, Waldburger & Susskind (1994) found that all but two positions in region 2.3 of s70 believed important for open complex formation, were tolerant to nonconservative amino acid substitutions, leaving open the question of how region 2.3 might participate in DNA unwinding. Though the contribution of region 2.3 to DNA melting is not yet under-

68 stood, all reported s subunit mutations to date that appear to affect isomerization to open complex lie within this region. We present here the ®rst evidence that conserved region 1 of E. coli s70 plays a role in the process of isomerization to the open complex (RPc1 to RPo) and the subsequent formation of the ternary initiated complex (RPinit). Region 1.1 is required for rapid isomerization from RPc1 to RPinit The presence of region 1.1 solely in primary s factors implies that it is important for the function of proteins within this de®ned subgroup of the s70 family. One role ascribed to this region is the modulation of s factor DNA binding activity, where region 1.1 acts to inhibit DNA binding in the absence of the core subunits (Dombroski et al., 1992, 1993). This ``inhibitory domain'' was roughly localized to within the ®rst 50 to 100 amino acids of region 1.1 (Dombroski et al., 1992, 1993). An intramolecular contact was proposed to account for the inhibition, in which region 1.1 interacts with region 4 of s70 (Dombroski et al., 1993). Here, analysis of the transcriptional activity of several truncated s70 polypeptides in vitro shows that region 1.1 of the N-terminal domain is also essential for effective transcription initiation. The ®rst detectable protein-DNA complex to form during transcription initiation is RPc1, followed by progression to RPc2 and RPo, and then transition to the ternary initiated complex, RPinit. Several lines of evidence argue that even the s derivatives with the largest truncations bind normally to DNA to form RPc1. First, the kinetic analysis of abortive initiation resulted in equivalent initial binding constants (KB) for holoenzymes containing FL,
50,
75, and
100 s factors. Secondly, DNase I footprinting experiments using these enzymes, as well as holoenzyme containing
133, result in protection by protein-DNA complexes over similar protein concentration ranges. Finally, in ®lter binding experiments, FL and
100 holoenzymes exhibit the same degree of ®lter retention of promoter DNA under low stringency wash conditions (0.1 M NaCl). Since the kinetics of initiation showed a marked reduction in the rate of isomerization (kf) by a factor of 20, and since we could detect no effect of the truncations on the ef®ciency of promoter clearance, we predicted that transitions among the various intermediate complexes between RPc1 and RPinit must be affected by the removal of region 1.1. Holoenzyme with
100 clearly progresses from RPc1 to RPc2, then to RPo, but at a much slower rate than FL holoenzyme. Time-dependent DNase I footprinting and KMnO4 modi®cation support this contention, since all of these complexes can be observed for
100, given enough time. Even though
100 eventually forms open complexes that appear equivalent in the extent of strand opening to FL, as judged by KMnO4 susceptibility, it was still unable to ef®ciently catalyze

70 Region 1 and Transcription Initiation

RNA synthesis to match FL, despite being given suf®cient time to form open complexes. We further demonstrate that
100 is additionally impaired in forming ternary initiated complexes, RPinit, by the instability of the
100 complexes to an 0.8 M NaCl wash. In summary, deletion of region 1.1 from s70 creates an RNA polymerase mutant that initially binds to promoter DNA to form the earliest closed complex, but is then signi®cantly hindered in performing the isomerizations necessary to ef®ciently progress through the intermediate complexes that precede processive RNA synthesis. These defects may in part be the indirect result of the removal of a large segment of the s polypeptide or, alternatively, could be directly due to the loss of the region. This is the ®rst report of any role for region 1.1 in transcription initiation. Region 1.2 is required for isomerization steps preceding the formation of RPo Holoenzyme formed with
133 is incapable of directing the synthesis of either abortive or run-off RNA products, and both DNase I and KMnO4 footprinting analysis show that
133 holoenzyme is arrested in a complex which most resembles the earliest detectable closed complex, RPc1. Thus, the deletion of region 1.2 (
133), in contrast to the effects of the
75 and
100 N-terminal deletions, appears to prevent RNA polymerase from progressing beyond the ®rst closed complex. Region 1.2 is present in all primary and alternative s factors examined to date, with the exception of Myxococcus xanthus SigB and Salmonella typhimurium FliA (Lonetto et al., 1992). These results suggest that region 1.2 may be required for the transitions to open complex formation, prompting the ®rst preliminary assignment of function to this highly conserved region. Participation of s factor in transcription initiation Although the mutants we have analyzed carry large deletions of the N terminus, they hint that regions 1.1 and 1.2 may participate sequentially in distinct phases of the isomerization mechanism, with region 1.2 directing the transition from RPc1 to RPc2 and region 1.1 enhancing the transition from RPc2 to RPo and RPinit. It is equally plausible that region 1.2 is required for both the RPc1 to RPc2 and the RPc2 to RPo transitions, with region 1.1 improving the overall ef®ciency of the process. In either case, these events may result from s factor intramolecular interactions, s factor-DNA interactions, or s factor-core interactions. Point mutations in regions 1.1 and 1.2 will be sought that behave similarly to the N-terminal truncations, to reduce the concern that large deletions may affect structure in an unpredictable manner. The progressive deletion of N-terminal amino acids from s70 results in a concomitant increase

70 Region 1 and Transcription Initiation

69

Figure 10. Alignment of nine primary bacterial s factors. Shaded residues represent similarity at 60% or more per position, calculated using the Dayhoff PAM250 scoring matrix, and boxed residues represent 75% or greater identity. The abbreviations denote the following bacterial species: Escherichia coli, Salmonella typhimurium, Buchnera aphidicola, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus, and Leptospira borgpetersenii. Of the aligned sequences shown, N. gonorrhoeae, L. borgpetersenii, and L. monocytogenes were acquired in the BLAST search but not the FASTA search.

in the DNA binding activity of free s factor (Dombroski et al., 1993), and a decrease in the ef®ciency of transcription initiation when the s subunit is associated with core, as shown here. One simple model that derives from these observations

depicts region 1.1 re-establishing the proposed interactions with the C-terminal DNA binding domain(s) to facilitate the formation of RPo and/ or RPinit during transcription initiation. Interaction between region 1.1 and the DNA binding do-

70 main(s) might also help to disrupt contact with the promoter elements upon promoter clearance. Sequence alignment and conservation A comparison of aligned amino acid sequences of region 1.1 from several primary s factors reveals conserved and potentially mechanistically important residues. Limited homology within region 1.1 was originally detected in an alignment of the protein sequences of Bacillus subtilus sA (s43) and E. coli s70 (Gribskov & Burgess, 1986). Later alignments, which included both primary and alternative s factors, showed relatively poor conservation of region 1.1 (Lonetto et al., 1992). By restricting our analysis to a pool of primary s sequences, we show signi®cant homology in region 1.1, including conserved elements which may prove diagnostic of the primary s family. The reduced isomerization rates of
75 and
100 in vitro may simply be due to the removal of highly conserved residues at positions 52, 53, 55 and 62 (Figure 10). An alternative explanation for the
75 and
100 defects derives from the overall acidity of region 1.1. We note that amino acids between 18 and 75 are 35% acidic. This segment contains the highest concentration of acidic residues in region 1.1. In analogy, s54 contains a highly acidic patch (36%) of residues, termed the acidic trimer repeat region (ATR), which has been shown to participate in DNA strand opening (Sasse-Dwight & Gralla, 1990; Wong & Gralla, 1992). Although s70 and s54 are otherwise structurally and functionally unrelated, the acidic patch present in both proteins could play a similar role in open complex formation. The
50 truncation which is functional in vitro, lacks nine out of the 20 acidic residues, while the
75 construction removes all 20 of the acidic residues in this region. Future experiments with s70 will address whether the overall acidity of region 1.1 or speci®c amino acids between residues 50 and 75 are important for open complex formation. The behavior of the
50 truncation raises important questions regarding the function of region 1.1.
50 transcribes as ef®ciently as full-length s70 in vitro. The inability of
50 to complement the temperature-sensitive (ts) growth defect of a strain carrying rpoD800 or to restore growth when chromosomal rpoD expression is shut off, implies that the deleted region, while dispensable for transcription in vitro, is important for the function and/or stability of s70 in vivo. Comparison of the aligned region 1.1 sequences suggests that the failure of
50 to complement rpoD800 may be due to the removal of highly conserved amino acids (Figure 10). The primary and alternative s factors, as well as the divergent members of the s54 family, all facilitate promoter recognition and transcription initiation, despite signi®cant variation in the size and domain composition of the s polypeptide. For example, among the s70 family members, the alternative s subgroup uniformly lacks region 1.1.

70 Region 1 and Transcription Initiation

Yet region 1.1 is highly conserved among the primary s factors, implying functional signi®cance. By further analyzing initiation by a variety of different s factors, we hope to understand potential variations in the mechanism of open complex formation and initiation.

Materials and Methods Materials Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs. Taq DNA polymerase was purchased from Fisher Scienti®c. [a-32P]GTP (3000 Ci/mmol) and [g-32P]ATP (3000Ci/mmol) were purchased from Amersham and NTPs were from Pharmacia. Puri®ed Core RNA polymerase was from Epicentre Technologies (Madison, WI). Buffer and gel components were obtained from Sigma or Fisher Scienti®c. Oligonucleotides were synthesized by either Bioserve Biotechnologies (Laurel, MD) or the UT Medical SchoolMolecular Genetics Core Facility (Houston, TX). Plasmid constructions Oligonucleotides (27 to 30 nucleotides in length) designed to incorporate KpnI and HindIII restriction sites at the 50 and 30 ends of the rpoD gene fragments, were used to amplify full-length (FL) rpoD and deletions that result in truncations of 50, 75, 100, and 133 amino acids from the N terminus of s70 (Figure 1 b). Insert DNA was generated by the polymerase chain reaction (PCR) using pJH62 (Hu & Gross, 1988) as the template. PCR reactions contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 50 pmol each primer, 25 ng template DNA, and 2.5 units of Fisher Taq DNA polymerase, in a 100 ml reaction volume. A Perkin Elmer DNA thermal cycler was programmed for 35 cycles with 95 C for denaturation, 55 C for annealing, and 72 C for extension, with a time of one minute for each segment. Inserts were ligated into the KpnI and HindIII sites of either pQE-31 (Qiagen, Inc.) or pQE-30T (pQE-30 modi®ed to contain a thrombin site). The resulting constructions contain a sixhistidine tag at the N terminus. Oligonucleotides containing 50 and 30 XhoI restriction sites were used to amplify DNA encoding the ®rst 100 amino acids of s70 (region 1.1). The DNA fragment was ligated into the XhoI site of pET15b (Novagen), also resulting in a six-histidine tag at the N terminus. Plasmid constructions containing FL,
50,
100, and
133 were maintained in E. coli strain XL1-Blue (Stratagene). The region 1.1 plasmid was transformed into E. coli BL21 (Novagen, Inc.). The plasmid encoding
75 was maintained in E. coli BL21 with lacIq provided on pKZ-115 (gift from W. Margolin). Oligonucleotide sequences are available upon request. Overproduction and purification of s derivatives Protein expression was induced by addition of IPTG to exponentially growing cultures of E. coli XL1-Blue or BL21, to ®nal concentrations of 1.0 mM and 2.0 mM for pET15b and pQE-31/pQE-30T constructions, respectively. After 3-4 hours of growth at 37 C, cells were harvested by centrifugation. 0.6 g cell pellet was resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and lysed by two passes through a Parr Instruments (Moline, IL) nitrogen cell dis-

70 Region 1 and Transcription Initiation ruption bomb. With the exception of region 1.1, which was puri®ed from the soluble fraction under non-denaturing conditions, all s protein derivatives fractionated with the cell pellet. Denaturation of protein in the pellets was performed in binding buffer/6 M urea for one hour, followed by batch-af®nity puri®cation on Ni2 ‡ -activated resin (Novagen, Inc.), according to the recommendations of the manufacturer. Puri®ed, denatured extracts were dialyzed against a series of reconstitution buffers (50 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol) containing, in sequence, 6 M, 4.5 M, 3 M, 1.5 M and no urea, followed by dialysis into storage buffer (50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 50 mM NaCl, 50% glycerol). Concentrations were determined by the Bio-Rad protein assay. Reconstitution of RNA Polymerase and transcription in vitro To reconstitute holoenzyme, the core subunits were mixed with a four- to sixfold molar excess of each truncated s and incubated on ice for 15 minutes. Optimal transcriptional activity of each s preparation was determined by using a series of ratios of s to core. For transcriptions with region 1.1 added in trans, the indicated molar excess of region 1.1 (Figures 6 and 7) was incubated with s for ten minutes on ice, followed by the addition of core and another ten minute incubation on ice. Template DNA fragments of 230 bp and 280 bp in length, containing the lPR and Ptac promoters and encoding 80 and 100-nucleotide transcripts, respectively, were generated by PCR, using primers ¯anking each promoter on template DNA, pBR81 (Suh et al., 1993) and M13mp19ptac (Dombroski et al., 1992). Sequences of the oligonucleotides are available upon request. All multi-round transcription reactions were performed in volumes of 15 ml. When quantifying total transcript formed in run-off transcription, template (8.8 nM, ®nal concentration) was added to an equimolar amount of reconstituted holoenzyme, in transcription buffer (100 mM KCl, 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.1 mM DTT), and incubated at 37 C for ten minutes. Reactions were initiated by the addition of 1.2 mCi [a-32P]GTP (3000 Ci/mmol) and NTPs to ®nal concentrations of 200 mM ATP, CTP, UTP and 20 mM GTP. After incubation for ten minutes at 37 C, the reactions were terminated by the addition of 5 ml of formamide stop solution (US Biochemical). To limit transcription to a singleround, heparin was added to a ®nal concentration of 50 mg/ml, together with the initiating NTPs, following preincubation at 37 C. Single-round transcription, using the ®nal concentration of holoenzyme established for multi-round assays, was performed with both equimolar and limiting template concentrations, with equivalent results. Single round reactions were ethanol precipitated to concentrate, prior to addition of formamide stop solution. Samples were heated for four minutes at 100 C and resolved on 8% (w/v) denaturing polyacrylamide gels. The same conditions were employed when quantifying total abortive transcript formed, except that 200 mM ApU (adenylyl [30 -50 ] uridine) was substituted for NTPs , to limit the product formed to a threenucleotide transcript, which was visualized on 24% denaturing polyacrylamide gels. Bands were quanti®ed directly using a Packard Instantimager. Kinetic experiments were conducted as follows. Reaction mixtures containing transcription buffer, limiting template (1.5 nM) , ApU and label (as above), were added to

71 holoenzyme, to a ®nal reaction volume of 110 ml. 10 ml sample aliquots were removed and stopped at speci®ed times, and the product resolved on 24% denaturing polyacrylamide gels. Bands were quanti®ed directly using a Packard Instantimager. Kinetic constants were calculated according to the methods of McClure (1980). DNase I cleavage analysis and KMnO4 modification The plasmid, pBR81, was digested with SpeI and the resulting fragments were 50 end-labeled using [g-32P]ATP (3000 Ci/mmol) and phage T4 polynucleotide kinase (New England Biolabs), followed by digestion with BssHII. The 237 bp labeled fragment containing the lPR promoter was then isolated from 2% metaphor agarose gels (FMC BioProducts) and puri®ed using the QIAquick gel extraction kit (Qiagen). Holoenzyme-promoter DNA complexes (1 pmol/ 0.1 pmol) were formed in 50 ml of DNase I buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 100 mg/mL BSA), in the presence of 0.2 mM ApU and GTP, for two and 40 minutes, at 37 C. DNase I (Promega) was used at 0.4 to 1 unit for 40 to 115 seconds at 37 C. Reactions in the presence of heparin were incubated for two and 40 minutes at 37 C, followed by addition of heparin (50 mg/ml ®nal concentration) and an additional ten minute incubation, prior to DNase I cleavage. Negative control footprinting analysis, following a 40 minute incubation, was performed with 6 pmol of s factor and 1 pmol of core, to maintain concentrations used in holoenzyme-promoter footprinting reactions. Reactions were stopped by addition of 50 ml phenol:chloroform. The aqueous phase was precipitated with ethanol. Samples were resuspended in loading dye (8 M urea, 0.5  TBE, 0.04% (w/v) bromophenol blue, 0.04% (w/v) xylene cyanol), heated for 45 seconds at 90 C and immediately applied to 8% denaturing polyacrylamide gels. Gels were electrophoresed at 2000 V for three hours, dried, and autoradiographed. KMnO4 modi®cation was performed as described by Newlands et al. (1991), using the DNase I buffer, as described above, with the ®nal concentration of DTT reduced to 0.2 mM. Time-dependent KMnO4 footprinting with region 1.1 added in trans was performed as follows. Template (1 to 5 nM) was preincubated in reaction buffer at 37 C and added to holoenzyme (reconstituted as in the transcription assays) with 10 to 20-fold molar excess of holoenzyme to template. 50 ml aliquots were removed at designated time points, treated with KMnO4, quenched, precipitated and washed as above. Modi®ed DNA was cleaved with 1 M piperidine (Newlands et al., 1991). Piperidine cleavage products were resolved on 8% denaturing polyacrylamide gels, dried and quanti®ed directly using a Packard Instantimager. Core competition assays were conducted by reconstituting a ®xed amount of core (20 nM) with a mixture containing equimolar concentrations of FL and either
100 or
133 s factor derivatives (80 nM of each), followed by incubation with a limiting amount of template (2 nM). After two minutes at 37 C, when
100 and
133 defects are known to be most severe, reactions were treated with KMnO4 and processed as above. The fraction of DNA cleaved in reactions containing
100 and
133 holoenzymes alone was normalized to the fraction cleaved in reactions with FL holoenzyme.
100 complexes exhibited 23% of the total cleavage present in FL-DNA complexes, while
133 complexes exhibited 7% of the cleavage present in the FL-DNA complexes. The prediction was made that 50% of the strand opening in a 1:1 mixed s assay would re-

72 ¯ect FL activity, while 50% would re¯ect either
100 or
133 levels of activity. So the expected fraction cleaved for (FL ‡
100) is [(0.5)(1) ‡ (0.5)(0.23)], or 62%, while the expected fraction cleaved for (FL ‡
133) is [(0.5)(1) ‡ (0.5)(0.07)], or 54%. Nucleotide binding stabilization assay The nucleotide binding stabilization assay was performed as described by Roe et al. (1984). Holoenzymepromoter DNA complexes were allowed to form for 40 minutes at 37 C, in binding buffer (40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 100 mg/ml BSA) containing 1 nM polymerase and 0.1 nM template. After 40 minutes, complexes were exposed to 0.1 mM ApU and GTP for 30 seconds. Reactions were then split into two aliquots and ®ltered through nitrocellulose ®lter disks (GS 0.22 mm; Millipore). Filters were rinsed with wash buffer (10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA) containing either 0.1 M NaCl (low stringency) or 0.8 M NaCl (high stringency). Washed ®lters were dried under an infrared heat source and subjected to liquid scintillation counting. Complementation of the rpoD800 mutation in vivo E. coli strain P90A5c (Calendar et. al., 1988) carrying the temperature-sensitive (ts) rpoD800 allele was transduced with P1Vir (srl W3110::Tn10) (gift from M. Winkler) to introduce a mutant recA allele to the chromosome, and conjugated with E. coli MH 7295, to introduce lacIq in trans on an F0 episome. Each s70 construction and the vector plasmid, pQE-30T, were transformed into this P90A5c derivative strain. The transformants were incubated at 30 C in liquid LB medium, diluted serially, spread on LB plates in the absence of IPTG and scored for the ability to complement the rpoD800 ts growth defect at 44 C. Expression of s 70 derivatives in E. coli strain CAG 20176 Plasmid DNA encoding each s70 derivative, as well as the parent vector, pQE-30T, was transformed into E. coli strain CAG 20176 ([ Cam]Ptrp-rpoD zgh::Tn10) (gift from C. Gross), an MC1061 (Meissner et al., 1987) derivative, in which a chloramphenicol cassette, followed by a synthetic trp promoter lacking the trp attenuator, is inserted between the dnaG and rpoD genes on the chromosome. rpoD expression is controlled by the trp repressor. Growth in LB medium requires 0.2 mM indole-3-acrylic acid (IAA) (ICN). Transformants were recovered in LB (supplemented with 2% (w/v) glucose and 0.2 mM IAA). Single colony transformants were picked and streaked onto LB (supplemented with 0.2 mM IAA, and 100 mg/ml Ampicillin, 10 mg/ml tetracycline). Single colonies were then resuspended in 60 ml of 1  M9 salts, and 25 ml aliquots of the suspension were used to inoculate 2 ml each of LB ‡/ÿ IAA (0.2 mM). Cultures were incubated at 37 C and the A600 was determined at midlog phase. Growth in the absence of IAA implied that the transformed s construction could complement in the absence of expressed chromosomal rpoD. Sequence analysis Preliminary sequence analysis was carried out using the Wisconsin Package, Version 8-UNIX (1994), Genetics

70 Region 1 and Transcription Initiation Computer Group (GCG) software. FASTA analysis, with a gap creation penalty of 12.0 and a gap extension penalty of 4.0, was ®rst conducted, using the ®rst 60 amino acids of E. coli s70 as the query sequence. The same query sequence was then entered into a BLAST search (Altschul et al., 1990), performed through the National Center for Biotechnology Information (NCBI). The alignment was calculated using Pileup, a GCG program which optimizes pairwise sequence alignments. The gap creation and extension penalties for this Pileup analysis were 3.00 and 0.10, respectively. The sequence alignment was then imported into SeqVu 1.0.1 for editing and display (Garvan Institute of Medical Research 1992-1995).

Acknowledgements We thank D. Le, B. Johnson, and O. Lee for technical assistance, Dr L. Hsu for helpful advice on abortive initiation, and Drs C. Gross and M. Lonetto for strain CAG 20176, and Dr C. Gross for helpful discussions. We are grateful to B. Johnson and Drs W. Margolin, S. Kaplan and J. Jones for critical reading of the manuscript. This study was supported by Grant-in-Aid 94G-263 from the Texas Af®liate of the American Heart Association and by research grant NP-902 from the American Cancer Society, both to A.J.D.

References Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment research tool. J. Mol. Biol. 215, 403±410. Bernard, S. L. & Meares, C. F. (1986). The s subunit of RNA polymerase contacts the leading ends of transcripts 9-13 bases long on the l PR promoter but not on T7 A1. Biochemistry, 25, 5914± 5919. Buckle, M., Geiselman, J., Kolb, A. & Buc, H. (1991). Protein-DNA cross-linking at the lac promoter. Nucl. Acids Res. 19, 833 ±840. Calendar, R., Erickson, J. W., Halling, C. & Nolte, A. (1988). Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional s32. J. Bacteriol. 170, 3479± 3484. Craig, M. L., Suh, W. & Record, M. T., Jr (1995). HO. and DNase I probing of Es70 RNA polymerase-l PR promoter open complexes: Mg2 ‡ binding and its structural consequences at the transcription start site. Biochemistry, 34, 15624± 15632. Daniels, D., Zuber, R. & Losick, R. (1990). Two amino acids in an RNA polymerase s factor involved in recognition of adjacent base pairs in the ÿ10 region of a cognate promoter. Proc. Natl Acad. Sci. USA, 87, 8075± 8079. DeHaseth, P. L. & Helmann, J. D. (1995). Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA. Mol. Microbiol. 16, 817± 824. Dombroski, A. J., Walter, W. A., Record, M. T., Jr, Siegele, D. A. & Gross, C. A. (1992). Polypeptides containing highly conserved regions of transcription initiation factor s70 exhibit speci®city of binding to promoter DNA. Cell, 70, 501± 512.

70 Region 1 and Transcription Initiation Dombroski, A. J., Walter, W. A. & Gross, C. A. (1993). Amino-terminal amino acids modulate s-factor DNA-binding activity. Genes Dev. 7, 2446± 2455. Gardella, T., Moyle, H. & Susskind, M. M. (1989). A mutant Escherichia coli s70 subunit of RNA polymerase with altered promoter speci®city. J. Mol. Biol. 206, 579± 590. Gribskov, M. & Burgess, R. R. (1986). Sigma factors from E. coli, B. subtilis, phage SPO1, and phage T4 are homologous proteins. Nucl. Acids Res. 14, 6745± 6763. Gross, C. A., Lonetto, M. & Losick, R. (1992). Sigma factors. In Transcriptional Regulation (McKnight, S. L. & Yamamoto, K. R., eds), vol. 1, pp. 129± 176, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Hawley, D. K. & McClure, W. R. (1980). In vitro comparison of initiation properties of bacteriophage l wild-type PR and 3 mutant promoters. Proc. Natl Acad. Sci. USA, 77, 6381± 6385. Helmann, J. D. & Chamberlin, M. J. (1988). Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 57, 839 ± 872. Hinkle, D. C. & Chamberlin, M. (1970). The role of sigma subunit in template site selection by E. coli RNA polymerase. Cold Spring Harbor Symp. Quant. Biol. 35, 65±72. Hsu, L. M. (1996). Quantitative parameters for promoter clearance. Methods Enzymol. 273, 59± 71. Hu, J. C. & Gross, C. A. (1988). Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli k-12. J. Mol. Biol. 203, 15± 27. Jones, C. H. & Moran, C. P., Jr (1992). Mutant s factor blocks transition between promoter binding and initiation of transcription. Proc. Natl Acad. Sci. USA, 89, 1958± 1962. Jones, C. H., Tatti, K. M. & Moran, C. P., Jr (1992). Effects of amino acid substitutions in the ÿ10 binding region of sE from Bacillus subtilis. J Bacteriol. 174, 6815± 6821. Juang, Y. & Helmann, J. D. (1994). A promoter melting region in the primary s factor of Bacillus subtilis. J. Mol. Bio. 235, 1470± 1488. Lesley, S. A. & Burgess, R. R. (1989). Characterization of the Escherichia coli transcription factor s70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry, 28, 7728± 7734. Lonetto, M., Gribskov, M. & Gross, C. (1992). MiniReview: The s70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174, 3843± 3849. Losick, R. & Pero, J. (1981). Cascades of sigma factors. Cell, 25, 582± 584. McClure, W. R. (1980). Rate-limiting steps in RNA chain initiation. Proc. Natl Acad. Sci. USA, 77, 5634± 5638. Meissner, P. S., Sisk, W. P. & Berman, M. L. (1987). Bacteriophage l cloning system for the construction of directional cDNA libraries. Proc. Natl Acad. Sci. USA, 84, 4171. Melancon, P., Burgess, R. R. & Record, M. T., Jr (1982). Nitrocellulose ®lter binding studies of the interactions of E. coli RNA polymerase holoenzyme with deoxyribonucleic acid restriction fragments: evidence for multiple classes of non-promoter interactions, some of which display promoter-like properties. Biochemistry, 21, 4318± 4331. Merrick, M. J. (1993). In a class of its own-The RNA polymerase sigma factor s54 (sigma N). Mol. Microbiol. 10, 903±909.

73 Newlands, J. T., Ross, W., Gosink, K. K. & Gourse, R. L. (1991). Factor-independent activation of Escherichia coli rRNA Transcription. J. Mol. Bio. 220, 569 ± 583. Record, M. T., Jr, Reznikoff, W. S., Craig, M. L., McQuade, K. L. & Schlax, P. J. (1996). Escherichia coli RNA polymerase (Es70), promoters, and the kinetics of the steps of transcription initiation. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., ed.), vol. 1, pp. 792± 820, ASM Press, Washington DC. Roe, J. & Record, M. T., Jr (1985). Regulation of the kinetics of the interaction of Escherichia coli RNA polymerase with the lPR promoter by salt concentration. Biochemistry, 24, 4721± 4726. Roe, J., Burgess, R. R. & Record, M. T., Jr (1984). Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lPR promoter. J. Mol. Biol. 176, 495± 521. Rong, J. C. & Helmann, J. D. (1994). Genetic and physiological studies of Bacillus subtilis sA mutants defective in promoter melting. J. Bacteriol. 176, 5218± 5224. Sasse-Dwight, S. & Gralla, J. D. (1990). Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor s54. Cell, 62, 945± 954. Saucier, J. & Wang, J. (1972). Angular alteration of the DNA helix by E. coli RNA polymerase. Nat. New Biol. 239, 167± 170. Siegele, D. A., Hu, J. C., Walter, W. A. & Gross, C. A. (1989). Altered promoter recognition by mutant forms of the s70 subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206, 591±603. Simpson, R. B. (1979). The molecular topography of RNA polymerase-promoter interaction. Cell, 18, 277± 285. Stragier, P., Parsot, C. & Bouvier, J. (1985). Two functional domains conserved in major and alternate bacterial sigma factors. FEBS Letters, 187, 11± 15. Suh, W. C., Leirmo, S. & Record, M. T., Jr (1992). Roles of Mg2 ‡ in the mechanism of open complex formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lPR promoter: evidence for a second open complex requiring Mg2 ‡ . Biochemistry, 31, 7815± 7825. Suh, W. C., Ross, W. & Record, M. T., Jr (1993). Two open complexes and a requirement for Mg2 ‡ to open the lPR transcription start site. Science, 259, 358± 361. Taylor, W. E. & Burgess, R. R. (1979). Escherichia coli RNA polymerase binding and initiation of transcription on fragments of lrifd 18 DNA containing promoters for l genes and for rrnB, tufB, rplK, A, rplJ, L, and rpoB, C genes. Gene, 6, 331± 365. Travers, A. A. & Burgess, R. R. (1969). Cyclic re-use of the RNA polymerase sigma factor. Nature, 222, 537± 540. Waldburger, C. & Susskind, M. M. (1994). Probing the informational content of Escherichia coli s70 region 2.3 by combinatorial cassette mutagenesis. J. Mol. Biol. 235, 1489± 1500. Waldburger, C., Gardella, T., Wong, R. & Susskind, M. M. (1990). Changes in conserved region 2 of Escherichia coli s70 affecting promoter recognition. J. Mol. Biol. 215, 267± 276. Wong, C. & Gralla, J. D. (1992). A role for the acidic trimer repeat region of transcription factor s54 in setting the rate and temperature dependence of promoter melting in vivo. J. Biol. Chem. 267, 24762± 24768.

74

70 Region 1 and Transcription Initiation

Zuber, P., Healy, J., Carter, H. L., III, Cutting, S., Moran, C. P., Jr & Losick, R. (1989). Mutation changing the

speci®city of an RNA polymerase sigma factor. J. Mol. Biol. 206, 605± 614.

Edited by M. Gottesman (Received 23 September 1996; received in revised form 23 December 1996; accepted 2 January 1997)