Promoter recognition and discrimination by EsS RNApolymerase

Tamas Gaal,1 Wilma Ross,1 Shawn T. Estrem,1 Lam H.

Nguyen,2 Richard R. Burgess2 and Richard L.

Gourse1*1Department of Bacteriology, University of Wisconsin,

Madison WI 53706, USA.2Department of Oncology, University of Wisconsin,

McArdle Lab, Madison WI 53706, USA.

Summary

Although more than 30 Escherichia coli promoters

utilize the RNA polymerase holoenzyme containing sS

(EsS), and it is known that there is some overlap

between the promoters recognized by EsS and by the

major E. coli holoenzyme (Es70), the sequence

elements responsible for promoter recognition by

EsS are not well understood. To define the DNA

sequences recognized best by EsS in vitro, we started

with random DNA and enriched for EsS promoter

sequences by multiple cycles of binding and selec-

tion. Surprisingly, the sequences selected by EsS

contained the known consensus elements (210 and

235 hexamers) for recognition by Es70. Using genetic

and biochemical approaches, we show that EsS and

Es70 do not achieve specificity through ‘best fit’ to

different consensus promoter hexamers, the way that

other forms of holoenzyme limit transcription to

discrete sets of promoters. Rather, we suggest that

EsS-specific promoters have sequences that differ

significantly from the consensus in at least one of the

recognition hexamers, and that promoter discrimi-

nation against Es70 is achieved, at least in part, by the

two enzymes tolerating different deviations from

consensus. DNA recognition by EsS versus Es70

thus presents an alternative solution to the problem of

promoter selectivity.

Introduction

Escherichia coli RNA polymerase (RNAP) consists of b,

b’, s, v and two a subunits. The core enzyme (a2bb’v) is

capable of transcription elongation, whereas s is required

for specific promoter recognition and transcription

initiation. Escherichia coli has seven different s subunits:

s70 (encoded by rpoD ), s54 (rpoN ), s32 (rpoH ), sS (rpoS ),

sE (rpoE ), s28 (rpoF ) and sFecI ( fecI ), each responsible

for directing RNAP to a different set of promoters, and, in

so doing, changing the pattern of gene expression (Gross

et al., 1998; Ishihama, 2000).

Es70, the holoenzyme responsible for transcription of

the majority of genes in E. coli, recognizes three DNA

sequence elements (all sequences provided below are for

the non-template strand, 50 to 30). The 210 element,

consensus TATAAT, is centred approximately 10 bp

preceding the transcription start site and is recognized

by regions 2.3 and 2.4 of s70. The 235 element,

consensus TTGACA, is 16–19 bp upstream from the

210 element and interacts with region 4.2 of s70 (Gross

et al., 1998). The UP element, an AT-rich region upstream

of the 235 hexamer, is recognized by the C-terminal

domains of the a subunits (Ross et al., 1993; Blatter et al.,

1994). In addition, some Es70 promoters contain an

extension of the 210 hexamer, TGTGn, immediately

upstream of the 210 motif, that is recognized by region 2.5

of s70 (Barne et al., 1997; Burr et al., 2000). Generally, the

more of these elements present in a particular promoter

and the more similar their sequences are to the consensus

sequences, the better the binding by RNAP (Gross et al.,

1998; Ross et al., 1998).

The synthesis, accumulation, and activity of sS (also

referred to as s38 or KatF) are controlled by a number of

positive and negative regulatory systems (Hengge-Aronis,

1996a; Ishihama, 2000). As a result, sS accumulates in the

cell at the beginning of stationary phase and directs RNAP

to transcribe stationary phase-specific genes (Mulvey and

Loewen, 1989; Nguyen et al., 1993). In addition, the

presence of specific transcription factors, high intracellular

osmolarity, and/or negative supercoiling can favour

transcription by EsS at certain promoters (Hengge-Aronis

et al., 1991; Kusano et al., 1996; Hengge-Aronis, 1996b;

Ishihama, 2000). As sS is involved in responses to osmotic

and oxidative stress, it is sometimes considered a master

regulator of stress responses (Hengge-Aronis, 1996b).

EsS is also required for virulence in a number of

pathogenic bacteria (Beltrametti et al., 1999; Suh et al.,

1999).

Although promoters recognized by EsS have been

compiled, they do not display a distinctive consensus

sequence (Espinosa-Urgel et al., 1996; Ishihama, 2000).

It has been suggested that EsS recognizes 210

elements similar, but not identical, to the consensusAccepted 6 September, 2000. *For correspondence. E-mail rgourse@bact.wisc.edu; Tel. (11) 608 262 9813; Fax (11) 608 262 9865.

Molecular Microbiology (2001) 42(4), 939–954

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recognized by Es70 (Espinosa-Urgel et al., 1996; Lee

and Gralla, 2001). It has also been suggested that a

235 consensus sequence for EsS, if it exists at all, is

different from the consensus element utilized by Es70,

for example, curved DNA with no specific 235 sequence

(Espinosa-Urgel et al., 1996), CC instead of TT at the

upstream end of the 235 element (Wise et al., 1996) or

CTGCAA as a 235 element (Bohannon et al., 1991;

Vicente et al., 1991; Ballesteros et al., 1998). Therefore,

the consensus DNA sequence for recognition by EsS is

still unclear.

We chose an in vitro approach to determine the

sequences recognized best by EsS, reasoning that

understanding the biochemical behaviour of the enzyme

in the absence of other factors, and then integrating these

data with the extensive information about EsS obtained in

vivo, might facilitate understanding of the behaviour of the

enzyme in the complex environment of the cell. We

generated a DNA library containing every possible

sequence variant in the s recognition regions and used

EsS to select those sequences that bound preferentially in

vitro. The 210 and 235 hexamers obtained were identical

to those recognized by Es70, raising an interesting

problem about promoter selectivity of the two holoen-

zymes. Based on genetic and biochemical approaches,

we show that the two forms of holoenzyme prefer the same

consensus hexamers for transcription, and that specificity

can be achieved by differential tolerance of the enzyme for

deviations from the consensus. This model is consistent

with the observation that many stationary phase-specific

genes are transcribed in vitro by both Es70 and EsS

(Tanaka et al., 1993; Tanaka et al., 1995), and that sS

shows considerable sequence homology to s70 in the

regions responsible for specific promoter recognition, 2.3,

2.4, 2.5 and 4.2 (Lonetto et al., 1992), but it differs

conceptually from the standard model for promoter

recognition in which each holoenzyme has its own discrete

DNA recognition elements responsible for transcribing

discrete sets of promoters.

Results

Rationale and experimental design

Footprinting experiments with an EsS-specific promoter,

bolA1, indicated previously that EsS protects about 70 bp

of promoter sequence (from about 257 to 117), similar in

length and position to that protected by Es70 (Nguyen and

Burgess, 1997). Based on this, and on the similarity in the

amino acid sequences of the two s factors in the regions

responsible for DNA binding (regions 2.3–2.5, and 4.2),

we decided to sample a population of DNA sequences

containing all possible sequences from 238 to 12.

The number of potential variants for 40 bp of DNA is

about 440 (¼1024) different DNA sequences, far too many

to test in vivo. Therefore, to test all possible recognition

sequences for optimal EsS binding, we used an in vitro

selection procedure with DNA fragments containing

randomized promoter sequences. Similar approaches

have been utilized previously to identify optimal binding

sequences for numerous nucleic acid binding proteins

(Blackwell and Weintraub, 1990; Pollock and Treisman,

1990; Tuerk and Gold, 1990; Wright et al., 1991; Estrem

et al., 1998; 1999).

One copy of each possible 40 bp sequence variant

would generate about 1 kg of DNA, far exceeding what can

be manipulated experimentally even in vitro. Therefore, we

performed the in vitro selection in two steps, randomizing a

20 bp segment to generate libraries containing at least one

copy of 420 (,1012) different sequences in each step. In

the first step, a selection was performed with promoter

fragments randomized from 218 to 12. This was followed

by a second selection with promoter fragments random-

ized from 238 to 219 in the context of the selected 218 to

12 sequence.

Highly purified EsS, containing no detectable s70 or

other s factors (Nguyen et al., 1993), was added to the

library of DNA fragments under limiting conditions so that

only 0.5–5% of the DNA fragments formed complexes

with RNAP. After separation of the complexes from free

DNA on a non-denaturing polyacrylamide gel, DNA from

the bound fraction was eluted, PCR-amplified and the

procedure was repeated for multiple cycles to enrich for

the best binding sequences (Fig. 1A). As EsS-DNA

complexes migrated to a location different from

Es70-DNA complexes (Fig. 1B), contamination with DNA

fragments bound by undetectable levels of Es70 (if

present) would be minimized at each cycle in the

enrichment. We note that our binding assay selects for

sequences resulting in the maximal rate of open complex

formation, but these sequences need not be optimal for

Fig. 1. A. Steps in the in vitro selection process. See text for details.B. Electrophoretic mobility-shift illustrating that EsS-promotercomplexes migrate to a different position than EsS-promotercomplexes containing the same 32P-labelled DNA fragment.

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each step leading to individual intermediates in the

transcription initiation pathway.

Selection of the 210 promoter region by EsS

To identify EsS promoter determinants in the region

containing the 210 element, EsS was incubated with a

promoter fragment library containing randomized

sequences from 218 to 12 and bolA1 promoter

sequences from 254 to 219 and from 13 to 116 (210

rnd; Fig. 2A). After 17 cycles of selection by EsS, 16 DNA

fragments were cloned and sequenced (see Experimental

procedures ). A close match to the 11 bp sequence

TGTGCTATA(C/A)T was present on each of 12 promoter

fragments (Fig. 2B and C; the other four fragments

contained large deletions and probably were selected for

artifactual reasons described in Experimental pro-

cedures ). The 11-mer sequence (e.g. in 210 sel 1)

showed limited similarity to the bolA1 promoter but

contained the Es70-like 210 hexamers, TATAAT or

TATACT (Fig. 2). The 11-mer also contained the con-

sensus ‘extended 2100 motif TGTG, that is found

upstream of the 210 hexamer in a subset of E. coli

promoters (Burr et al., 2000), and a C between the

extended 210 motif and the 210 hexamer. The other 9 bp

of the randomized region differed among the selected

promoter fragments, indicating the promoters were of

independent origin.

The position of the selected 11-mer was relatively

constant within the randomized region (from 217 to 27 in

eight cases, from 216 to 26 in three cases, and from 218

to 28 in one case). These data suggest that sequences

outside the randomized region contribute to positioning

EsS on the DNA and, therefore, have an important role in

recognition by EsS.

Selection of the 235 promoter region by EsS

To identify promoter determinants for EsS binding

upstream of 218, a second selection was performed

with a fragment library (235 rnd; Fig. 3A) containing

randomized basepairs from 238 to 219. Flanking

sequences in the starting population derived from the

210 sel #1 promoter (218 to 116; Fig. 2) and from the

Fig. 2. 210 region selection.A. Non-template strand DNA sequences of the bolA1 promoter, the starting DNA population (210 rnd ) and 210 sel #1 [the first sequence in (B)]. Therandomized region in 210 rnd (218 to 1 2) is indicated by ‘NNN…’. The Es70 consensus elements (Gross et al., 1998; Burr et al., 2000) areindicated.B. Sequences from the randomized region of 12 DNA fragments cloned after 17 cycles of selection with EsS in vitro. Sequences were aligned byidentities in the 210 region.C. Frequency of bases at each position in the 12 cloned promoters from (B). Numbers in bold indicate positions at which the same base was found inat least 10 out of the 12 promoters or at which either of two bases were found in all 12 promoters.

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‘SUB’ promoter (254 to 239; Rao et al., 1994). The 254 to

239 SUB sequence does not contain RNAP recognition

motifs (Rao et al., 1994) and was introduced to eliminate

interference from 235-like sequences upstream of

position 238 in bolA1 (Nguyen et al., 1993).

Twenty-two promoter fragments were cloned and

sequenced after 17 cycles of selection. Nineteen promoter

fragments of the correct length contained the sequence

CTTGACA from positions 236 to 230, exactly 17 bp

upstream from the 210 hexamer (Fig. 3B and C); the other

three fragments contained large insertions and probably

were selected for artifactual reasons, as described in

Experimental procedures ). In addition, there was a strong

preference for A residues at positions 229 and 228 and a

lesser preference for T at 227. Positions 238, 237 and

226 to 219 were different from clone to clone, confirming

that the selected promoters were of independent origin.

The selected 235 promoter region showed no similarity to

the same region of the bolA1 promoter, but it contained the

consensus 235 hexamer for recognition by Es70.

Characterization of the selected promoters in vitro

To analyse the effects of the selected sequences on

RNAP binding and transcription, promoters were con-

structed containing the selected 238 to 219 and 218 to

12 regions (either individually or combined together). To

facilitate direct comparison with the bolA1 promoter, each

construct contained sequences from bolA1 at all positions

outside the randomized regions (Fig. 4A). For simplicity,

these constructs are referred to as the ‘in vitro selected’

promoters, including 210 con (containing the selected

218 to 1 2 region from 210 sel #1; Fig. 2), 235 con

(containing the selected 238 to 219 region from 235 sel

#1; Fig. 3) and full con (containing both selected regions).

To verify that the selection resulted in DNA sequences

Fig. 3. 235 region selection.A. Non-template strand DNA sequences of the starting DNA population (235 rnd ) and 235 sel #1 [the first sequence in (B)]. The starting population(235 rnd ) contained the ‘SUB’ upstream sequence (254 to 219; Rao et al., 1994) and the 210 region from 210 sel #1. Randomized region (238 to219) is indicated by ‘NNN…’. The Es70 consensus elements (Gross et al., 1998; Burr et al., 2000) are indicated.B. Sequences from the randomized region of 19 DNA fragments after 17 cycles of selection with EsS in vitro.C. Frequency of bases at each position in the 19 cloned promoters from (B). Numbers in bold indicate positions at which the same base was found inat least 16 out of the 19 fragments.

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that bound EsS better than bolA1, we compared the

affinities of the DNA fragments for EsS under conditions

identical to those used for the selection (see Experimental

procedures ). The three selected promoters bound EsS

substantially better than bolA1 (data not shown), confirming

that the selection worked as expected. The relative order of

binding was full con . 235 con . 210 con .. bolA1.

To assess whether the increase in EsS binding by the

selected promoters relative to bolA1 resulted in increased

transcription, the activities of the three selected promoters

were compared with the activity of the bolA1 promoter in vitro.

When transcribed by EsS, all three selected promoters were

much more active than bolA1, and the 235 con promoter was

the strongest, in buffers containing 100–800 mM K-gluta-

mate or 10–400 mM KCl (Fig. 4B and data not shown). The

difference in activity between bolA1 and the selected

promoters was less dramatic at high RNAP concentrations

(data not shown). As the selected promoters also contained

the known consensus sequences for Es70 recognition, we

tested their transcription by Es70 in vitro under the same

conditions used for EsS. The selected promoters were

transcribed very efficiently by Es70, whereas transcription

from the bolA1 promoter was hardly detectable (Fig. 4C).

The selected promoters were transcribed similarly by

both holoenzymes in buffers containing 10–400 mM KCl

(Fig. 4D and data not shown) or 100–800 mM K-glutamate

(Fig. 4E and data not shown). In no case were the

promoters transcribed more than about twofold better by

EsS holoenzyme than by Es70. Therefore, in contrast to

the situation for some osmotically regulated promoters that

are transcribed only by EsS at high concentrations of

K-glutamate (Ding et al., 1995; Kusano and Ishihama,

Fig. 4. In vitro transcription.A. DNA sequences (254 to 116) of the promoters analysed. Sequences outside of the in vitro selected regions are identical in all four promoters andderive from bolA1. The fragments were inserted into a plasmid that contains a transcription terminator ,170 bp downstream of the transcription startsite (11).B. Transcription from the four promoters with EsS. The transcripts from the four promoters are indicated as ‘test’. The RNA I transcript from thevector is also indicated. Reactions contained Transcription buffer (see Experimental procedures ), 100 mM KCl and approximately 1 nM RNAP.C. Transcription from the same templates as in (B) but with Es70 instead of EsS. EsS and Es70 were assembled by addition of fivefold excess of s tothe same amount of core enzyme (see Experimental procedures ). The faint transcript noticeable above the test transcripts in the Es70 reactionsoriginates from a weak promoter whose putative 235 hexamer derives from bolA1 sequences about 10–13 bp upstream of the 235 hexamerresponsible for the major transcript (Nguyen et al., 1993).D. Transcription of the selected promoters at different KCl concentrations in vitro under otherwise identical reaction conditions as in (B) and (C). They-axis is the ratio of specific product (from either the 210 con, 235 con or full con promoter) resulting from transcription by EsS vs. Es70.E. Ratio of transcription by EsS to Es70 at different K-glutamate concentrations.

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1997), the selected promoters did not exhibit a large

preference for EsS at high osmolarity.

Because EsS and Es70 were prepared by adding

saturating amounts of s factor to aliquots of the same

preparation of core enzyme, it is valid to compare the

relative activities of EsS and Es70 on each of the

promoters (see Experimental procedures ). For each

selected promoter, the level of transcription by EsS and

Es70 was very similar, although the three selected

promoters differed in activity from each other. In contrast,

the wild-type bolA1 promoter was transcribed substantially

better by EsS than by Es70, whereas the plasmid-derived

RNA I promoter was transcribed much better by Es70 than

by EsS. We conclude that the in vitro selected promoters

are transcribed much better than the bolA1 promoter in

vitro, but they do not exhibit specificity for EsS over Es70.

Transcription from the full consensus promoter ( full

con ) was always weaker than from the promoter contain-

ing only the consensus 235 region, 235 con (Fig. 4B and

C). The full con promoter’s relative inactivity results from a

defect in promoter escape (T. Gaal, R.L. Gourse and

N. Shimamoto, unpublished).

Characterization of the in vitro selected promoters in vivo

We measured the activities of the bolA1, 210 con, 235

con and full con promoters as promoter– lacZ fusions to

determine whether the relative activities observed in vitro

were also observed in vivo, and also to determine whether

the promoters displayed the increase in promoter activity

in stationary phase characteristic of EsS-dependent

promoters (Fig. 5). The relative activities of the promoters

were consistent with the activities observed in vitro: the

235 con promoter was three to fivefold stronger than the

210 con or full con promoters, and all three were much

stronger than the bolA1 promoter, both in exponential and

in stationary phase (Fig. 5A and C).

The bolA1 promoter fragment was also introduced into a

lacZ fusion system that produces higher b-galactosidase

activity and lower background (‘System II’; Simons et al.,

1987; Rao et al., 1994), allowing a more accurate estimate of

transcription from weak promoters (see Fig. 5A and C inset).

bolA1 promoter activity increased about 18-fold in stationary

phase relative to exponential phase (Fig. 5B). In contrast,

there was less than a twofold increase in bolA1 promoter

activity in stationary phase in a strain lacking rpoS (Fig. 5D),

confirming previous reports that the bolA1 promoter is

EsS-dependent (e.g. Lange and Hengge-Aronis, 1991).

Although the 210 con, 235 con and full con promoters

were selected for binding to EsS, they were transcribed

efficiently even when EsS was not present (Fig. 5C), and

their activities increased only slightly in stationary phase

(Fig. 5D). As the 210 con, 235 con and full con promoters

are transcribed very efficiently by both EsS and Es70 in

vitro, and as the promoters contain the consensus

hexamers recognized by Es70, we conclude that these

promoters are transcribed by Es70 in exponential phase

and are probably transcribed by either EsS or Es70 (or

both) in stationary phase. [s70 is more abundant than sS,

even in stationary phase (Jishage et al., 1996)]. The

residual rpoS-independent small increase observed for all

four promoters in stationary phase could derive from

decreased competition for limiting Es70 when rRNA

transcription drops in slow or non-growing cells (see

Barker et al., 2001).

Interactions of EsS and Es70 RNAPs with specific

promoter positions

The results from the in vitro selections described above

Fig. 5. Transcription in vivo.A. The four promoters analysed in vitro (Fig. 4A) were fused to lacZ insingle copy on the chromosome, and b-galactosidase activities weremeasured in exponential and stationary phases as described inExperimental procedures. The bolA1 promoter was measured inSystem I (see Experimental procedures ) to allow direct comparisonwith the other System I promoter– lacZ fusions, but bolA1 was alsomeasured in System II (Inset, see Experimental procedures ) for moreprecise determination of the exponential/stationary phase ratio shownin (B). (C) and (D) were the same as (A) and (B) except the promoter–lacZ fusions were in an isogenic strain lacking rpoS.

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suggested that both EsS and Es70 prefer the same 210,

235 and extended 210 consensus sequences. However,

the identification of sequences in in vitro selection

experiments does not imply that each selected position

has the same relative importance to RNAP binding, as

even small increments to binding will result in selection,

nor do these experiments address whether EsS and Es70

interact with a particular promoter in the same manner. To

address whether the two enzymes recognize the same

promoter sequence differently, we used interference

footprinting, a technique in which DNA fragments with

modifications at different positions in the population can all

be assayed for protein binding at the same time.

Specifically, if a modification at a certain position interferes

with RNAP binding, the promoter fragment containing that

modification will be underrepresented in the bound

population. Interference footprinting thus allows estimation

of the relative contribution of individual positions in a

promoter to RNAP binding (although of course the effects

of every functional group or even every basepair are not

tested by any one interference probe).

We tested the effects of two different modifications that

alter the major groove surfaces of thymine or adenine in

DNA on binding by EsS and Es70. Uracil is identical to

thymine except it lacks the C5 methyl group in the major

groove (Devchand et al., 1993), whereas 7-deaza-7-nitro-

adenine (A*) introduces a bulky nitro group into the major

groove (Min et al., 1996). dUTP, or dA*, was incorporated

into promoter fragments at low frequency by polymerase

chain reaction (PCR) (Ross et al., 2001). The effects of

these substitutions on Es70 binding to the rrnB P1

promoter have been characterized previously (Ross

et al., 2001). Preliminary experiments suggested that

single base modifications in the full con promoter did not

interfere enough with binding by either holoenzyme to alter

the fraction bound in gel-shift assays (data not shown).

Therefore, the 235 con and rrnB P1 promoters were used

instead; these promoters bound both holoenzymes, but

Fig. 6. EsS and Es70 bind differently to thesame promoter sequence.A. Interference footprints of the 235 conpromoter with uracil substituted for thymine onaverage once per DNA fragment. The promoterfragment was labelled at the 30-end of the non-template strand. Signals result from strandcleavage only at the sites of uracil substitution.Scans are shown above the gel images (grey,no RNAP; blue, EsS-bound complex; red,Es70-bound complex). The 210 and 235hexamers are in upper case letters, and thethymine residues discussed in the text areindicated below the gel images.B. Interference footprints of the rrnB P1promoter with A* substituted for adenine onaverage once per DNA fragment. The promoterfragment was labelled at the 30-end of the non-template strand. Signals result from strandcleavage only at the sites of A* substitution.Colour coding used in the scans is the same asin (A). The adenine residues in the 210hexamer discussed in the text are indicatedbelow the gel images. For simplicity, theresidues in the 210 hexamer are numbered asin the 235 con promoter shown in (A). Becausethe distance between the transcription start siteand the 210 hexamer in rrnB P1 is 2 bp longerthan in 235 con, ‘211’ and ‘28’ are actually at213 and 210 in rrnB P1.

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with lower affinity than the full con promoter, so that uracil

or A* substitutions resulted in changes in the fraction of the

DNA population bound by RNAP.

Uracil incorporation at three non-template strand

positions in the 235 con promoter (one in the 210 region

at 212, and two in the 235 region at 234 and 235)

reduced binding by both EsS and Es70 (Fig. 6A). Uracil at

212 virtually eliminated binding by both enzymes. In

contrast, uracil at either 234 or 235 greatly reduced Es70

binding, but only moderately reduced EsS binding. Similar

differential effects of uracil substitution at 234 and 235 on

Es70 versus EsS binding were also observed on the rrnB

P1 promoter (data not shown). These results suggested

that even though both holoenzymes prefer the same

promoter sequence, they interact with that promoter

differently.

The effects of A* substitutions for adenine in the non-

template strand on Es70 and EsS binding to the rrnB P1

promoter are illustrated in Fig. 6B; qualitatively similar

results were obtained for the 210 con and 235 con

promoters (data not shown). Modification of either of two

positions in the 210 hexamer reduced RNAP binding,

whereas no effects were observed in the 235 region.

Binding of the two holoenzymes was affected differentially

at one of the 210 region positions, the highly conserved

A211 in the 210 hexamer. At this position, A* decreased

EsS binding by about 60% but decreased Es70 binding by

only about 30%. As with the uracil interference exper-

iments, the A* footprints indicate that EsS and Es70 bind

differently to the same promoter sequence. The less than

total inhibition observed from introduction of A* for the

highly conserved A211 could be attributable to the fact

that the N1 position on the base is most crucial for A211

function (Matlock and Heyduk, 2000), not the 6 and 7

positions on the base altered in A*.

A* substitution at A29 had a smaller effect than at

A211, and A* substitution at A28 had a larger effect than

at A211, but in both cases the effects were similar for the

two holoenzymes. The strong inhibition of RNAP binding

by A* at 28 could be attributable to steric clash by

introduction of the bulky nitro group, as typically mutations

at 28 have less severe effects on RNAP interactions than

at 211.

Basis for promoter selectivity by EsS versus Es70

The similarity in the intrinsic promoter recognition proper-

ties of EsS and Es70 raises the issue of how some

promoters could be almost entirely dependent on EsS.

That is, what accounts for some promoters being

transcribed only by EsS when both EsS and Es70 are

present in stationary phase, and for these same promoters

not being transcribed by Es70 in exponential phase? In this

section, we propose a model to address these questions

and then we provide experimental support for this

proposal.

Our data suggest that the preferred hexamer sequences

might be identical for EsS and Es70. However, naturally

occurring promoter sequences rarely match the consen-

sus perfectly for a particular RNAP. Differences from the

Es70 consensus sequence, in conjunction with the effects

of specific activators and repressors, are responsible for

differences in levels of transcription initiation from

individual Es70 promoters. The interference footprints

shown above indicate that certain modifications of the 210

and 235 hexamers affect binding by EsS and Es70

differently. Therefore, to explain differences in transcrip-

tion by EsS versus Es70, we suggest that non-preferred

basepairs in a promoter sequence might have different

effects on recognition by the two enzymes.

To test this general concept, we chose two positions in

the 235 element for detailed examination. The choice of

these positions was dictated by the results of our

interference footprinting studies (Fig. 6A) indicating that

the loss of the methyl groups at positions 234 and 235

affect discrimination by Es70 versus EsS and by previous

genetic evidence suggesting that C substitutions at 234

and 235 result in specificity for EsS (Wise et al., 1996;

Fig. 7. Substitution of CC for TT at the first two positions in the 235element differentially affects transcription by EsS versus Es70 both invivo and in vitro.A. The activity of the CC235 con promoter was measured inexponential (light bars) and stationary phase (dark bars) as describedin Fig. 5. Promoter activities (in Miller units) were measured frompromoter– lacZ fusions (System II, see Experimental procedures ) instrains containing or lacking rpoS. The CC235 con promoter is like235 con except that it has a CC substitution for TT at the first twopositions in the 235 hexamer. A promoter fragment with endpoints of240 to 12 was used to construct both the promoter– lacZ fusion andthe plasmid template for in vitro transcription. b-Galactosidaseactivities from the strains used in Fig. 7 (RLG5852, RLG5857) shouldnot be compared with those reported in Fig. 5, because the promotersassayed in Fig. 5 were in a different fusion system (see Experimentalprocedures ) and had different endpoints (254 to 116).B. Supercoiled plasmid pRLG5881containing the CC235 conpromoter (240 to 12) was transcribed with either EsS or Es70

(approx. 1 nM) in vitro as described in the legend to Fig. 4.

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Bordes et al., 2000). To confirm that the identities of 234

and 235 contribute to discrimination by Es70 versus EsS,

we constructed a CC-35 con promoter (i.e. a 235 con

promoter containing T-34C and T-35C substitutions) fused

to lacZ. The promoter DNA fragment used in this fusion

extended only to 240 to eliminate any contribution to

activity in vivo from a weak Es70-dependent promoter

upstream of the 235 hexamer that derives from bolA1

sequences (see Fig. 4C).

CC235 con was considerably weaker than 235 con and

displayed the characteristics of an EsS-specific promoter

in vivo (Fig. 7A). Transcription from CC235 con greatly

increased in stationary relative to exponential phase, and

this increase was almost completely dependent on the

presence of rpoS. (The promoter endpoints and the lacZ

reporter system used in Fig. 7 differ from those used in

Fig. 5. Thus, the absolute b-galactosidase activities in the

two figures should not be compared directly (see also

Experimental procedures ). We also tested transcription

from CC235 con with EsS and Es70 in vitro (Fig. 7B).

Consistent with the results obtained in vivo, CC235 con

was preferentially utilized by EsS in vitro. EsS tolerated the

CC substitution much better than did Es70 under every

condition tested.

To test whether EsS can tolerate only the CC

substitution at 234 and 235 or whether other substitutions

at these positions are tolerated as well in 235 con, we

generated all possible single and some two basepair

substitutions for the TT at 234 and 235, and we compared

transcription from each mutant by EsS in vitro to that from

235 con (Fig. 8A). None of the mutant promoters was

more active than 235 con, confirming that TT at these two

positions is optimal for recognition by EsS. None of the

substitutions decreased transcription by EsS more than

about twofold. CC235 con was transcribed by EsS worse

than most of these promoters, indicating that CCGACA

(Wise et al., 1996) is not the consensus 235 hexamer for

recognition by EsS. As the promoter with the single G

substitution at 235 was transcribed as well as 235 con in

vitro, but promoters with this substitution were not

obtained in the in vitro selection, it is possible that the in

vitro transcription assay is not sufficiently sensitive to

detect the slight preference for the consensus T at this

position, or there is an effect of promoter context in this

case.

In contrast to their small effects on transcription by EsS,

the TG, AA, CC, or GG substitutions decreased

transcription by Es70 as much as 10-fold, resulting in a

six to 10-fold preference for EsS over Es70 (Fig. 8B and C).

These results are consistent with the model that

discrimination between EsS and Es70 is accomplished

by differential tolerance of the two enzymes for specific

deviations from the consensus sequence.

Discussion

Es70 and EsS recognize the same consensus hexamer

sequences

We have studied the intrinsic promoter recognition

properties of EsS using an in vitro selection to identify

the DNA sequence determinants for EsS binding. In

contrast to expectations based on previous proposals (see

Fig. 8. Multiple substitutions at the first two positions in the 235element differentially affect transcription by EsS or Es70 in vitro.Supercoiled plasmids containing the 235 con promoter (TT atpositions 235 and 234) or the indicated variants at these positionswere transcribed with either EsS or Es70 (approx. 4 nM) in vitro asdescribed in the legend for Fig. 4 and Experimental procedures.Promoter activity is plotted relative to that of the 235 con promoter foreach enzyme. The 235 con promoter is transcribed similarly by EsS

and Es70 under these buffer conditions (see Fig. 4). Transcription wasmeasured at least three times from each promoter and is expressedrelative to transcription of 235 con promoter. Differences in therelative levels of transcription were less than 15% betweenexperiments.A. Transcription with EsS in vitro.B. Transcription with Es70 in vitro.C. Ratio of transcription with EsS/Es70 in vitro.

Promoter recognition and discrimination by Ess RNAP 947

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Introduction ), the binding sites selected by EsS contained

the consensus hexamer sequences for binding Es70, and

they initiated transcription at high levels by both

holoenzymes.

Many EsS-dependent promoters rely predominantly on

recognition of the 210 region (e.g. Tanaka et al., 1995;

Espinosa-Urgel et al., 1996; Colland et al., 1999; Lee and

Gralla, 2001). However, the results of the in vitro selection

and the extraordinary strength of the 235 con promoter,

relative to the bolA1 promoter, when transcribed by EsS in

vitro, suggest that the 235 element can also play a

prominent role in recognition by EsS in some promoter

contexts. In fact, some EsS-dependent promoters (e.g.

osmE; Bordes et al., 2000) have almost consensus 235

regions and appear to be strongly dependent on 235

hexamer interactions.

Differential tolerance of RNAP holoenzymes for deviations

from the same consensus hexamers

As both Es70 and EsS prefer the same consensus

hexamer sequences, there must be a mechanism for

preventing transcription from stationary phase-specific

promoters by Es70 in exponential phase. (Preventing

transcription from these promoters by EsS in exponential

phase is accomplished by limiting expression of sS.) We

suggest that holoenzyme specificity might rely, at least in

part (see below), on tolerance of the two enzymes for

different deviations from the same consensus sequences.

This hypothesis is supported by interference footprinting

studies showing that EsS and Es70 recognize the same

promoter sequence differently (Fig. 6) and by the effects of

certain promoter mutations constructed as a test of this

concept (Figs. 7 and 8). We found that, in the context of the

235 con promoter, substitutions of TT to TG, CC, AA, or

GG at the first two positions of the 235 hexamer resulted

in transcription in vitro that was strongly dependent on

EsS, in contrast to 235 con. These results are consistent

with genetic studies on the proU promoter, in which TT to

CC substitutions at the same positions strongly reduced

transcription and made it dependent on rpoS in vivo, and

on the osmY promoter, in which CC to TT substitutions

decreased discrimination between EsS and Es70 in vivo

(Wise et al., 1996).

We did not evaluate the relative preferences of EsS and

Es70 for each possible basepair at every position in the

consensus hexamers. However, several results strongly

suggest that, in addition to positions in the 235 hexamer,

there are positions in the 210 hexamer at which

interactions with the two RNAPs are differentially affected

by the same base modification or base substitution. These

include the results of our interference footprints (Fig. 6),

the fact that the promoter differing from bolA1 only in the

210 region (210 con ) is recognized by EsS but has lost its

EsS specificity (Fig. 4), and recent studies showing there is

differential binding of EsS and Es70 to fork-junction

templates with substitutions in the 210 region (Lee and

Gralla, 2001).

At position 28, A and C were about equally represented

in the sequences selected by EsS, whereas A is

considered the consensus for Es70-dependent promoters.

Although there are some differences between s70 and sS

in the region predicted to interact with position 28 (region

2.3; Lonetto et al., 1992; Malhotra et al., 1996; Fenton

et al., 2000), we suggest that the identity of 28 does not

play a major role in discrimination between the two

holoenzymes. We constructed promoters differing only by

A or C at this position and found they were transcribed

similarly by the two enzymes in vitro (data not shown), and

previous reports suggest that both Es70 and EsS tolerate A

or C at 28 (Oliphant and Struhl, 1988; Kolb et al., 1995;

Tanaka et al., 1995; Espinosa-Urgel et al., 1996).

Recognition of either A or C at 28 by both enzymes

could potentially be attributable to an interaction with the

same functional group on both bases, for example the

amino group on position 6 of A, or 4 of C.

Other potential contributions to differential promoter

recognition by EsS and Es70

Recent studies have proposed that a C at position 213

(i.e. the position just upstream from the 210 hexamer) is

preferred by EsS, and G213 is preferred by Es70 in certain

contexts, for example in the csiD and osmY promoters

(Becker and Hengge-Aronis, 2001) and in fork-junction

templates (Lee and Gralla, 2001). The results of our in vitro

selection strongly support the preference of EsS for C at

213. However, C213 is clearly not sufficient to

discriminate against recognition by Es70 in the context of

the in vitro selected promoters described here (Figs. 4 and

5). In addition, the rrn P1 promoters that are transcribed

much more efficiently by Es70 than by EsS (data not

shown) contain a C at this position.

Residues in addition to the consensus hexamers and

C-13 were selected by EsS in our in vitro binding

experiments, including a cytosine at the upstream flank

of the 235 hexamer (236), two adenines at the positions

just downstream of the 235 hexamer (229 and 228) and

the sequence TGTG at positions 214 to 217 (the

extended 210 region); 236, 229, and 228 are not

usually considered as being determinants of Es70 binding

and, therefore, it is formally possible that they might

contribute to specific EsS recognition. However, we

consider it improbable that these basepairs, by them-

selves, are responsible for discrimination between EsS

and Es70, as (i) C is also preferred by Es70 at 236 in the

lac and rrnB P1 promoters (Reznikoff, 1976; Josaitis et al.,

1990), and (ii) A to G substitutions for the A residues at

948 T. Gaal et al.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

229 and 228, at least in the context of the 235 con

promoter, did not affect activity substantially in vitro with

either holoenzyme (data not shown). [We note, however,

that an A-tract downstream of the 235 hexamer was

identified as a requirement for efficient rRNA promoter

activity by Chlamydia trachomatis RNAP (Tan et al.,

1998)]. The interaction between the extended 210 motif

(TGTG) and region 2.5 of s has been well documented as

an important determinant of both EsS- and Es70-depen-

dent transcription (Voskuil et al., 1995; Barne et al., 1997;

Becker et al., 1999; Colland et al., 1999; Burr et al., 2000).

A spacer length of 17 bp between the 210 and 235

hexamers was strongly favoured for recognition by EsS in

our in vitro selection. We found that Es70 tolerated a 16 bp

spacer in the context of the rrnB P1 promoter much better

than EsS, but a 16 bp spacer reduced the activity of the full

con promoter approximately the same for both holoen-

zymes (data not shown). Therefore, spacer-length could

contribute to EsS versus Es70 discrimination at some

promoters, but our preliminary studies indicate that the

effect of spacer length on the two holoenzymes is context-

dependent and complex.

Finally, our hypothesis does not exclude a role for

additional DNA-binding proteins in promoter discrimination

by EsS and Es70. That is, expression of some promoters

(although not those investigated here) is influenced by

differential effects of transcription factors or nucleoid-

associated proteins on the two holoenzymes (Arnqvist

et al., 1994; Bouvier et al., 1998; Colland et al., 2000).

s determinants for differential recognition of the same

consensus hexamers

The similarity between the EsS and Es70 consensus

sequences is consistent with the similarity of the motifs in

the two s factors responsible for promoter recognition.

Sixteen out of 28 amino acids in region 4.2 (residues 572–

599 of s70) and 25 out of 40 amino acids in regions 2.3 and

2.4 (residues 417–456 of s70) are identical in the two s

factors (Lonetto et al., 1992), including several residues

implicated in direct interactions with specific basepairs in

the 210 and 235 hexamers (e.g. Q437 in region 2.4, and

R584 and R588 in region 4.2; s70 numbering, Gross et al.,

1998).

Superimposed on the overall conservation between the

two s factors in the regions responsible for DNA binding

are discrete regions of divergence that could contribute to

promoter discrimination. Two clusters of four amino acids

in region 4.2 (residues 578–581 and 591–594 of s70) are

virtually identical among s70 members from different

species and among different sS members from different

species, but these sequences differ between s70 and sS

(Lonetto et al., 1992) and could potentially play a role in

discrimination between deviations from the consensus

235 hexamer. K173 of sS has been implicated in

recognition of C213 (Becker and Hengge-Aronis, 2001).

The corresponding residue in s70 is different (E458),

consistent with their different DNA recognition prefer-

ences. As interactions with the 210 element are complex,

involving binding to both single-stranded and double-

stranded DNA in multiple steps during the process of

transcription initiation, it is more difficult with present

information to predict specific amino acid residues in s70

and sS that might play a role in differential 210 region

recognition.

Promoters recognized by both EsS and Es70

The observed overlap in the sequences recognized by

EsS and Es70 potentially allows some promoters to be

transcribed by both holoenzymes under some conditions,

and yet to be transcribed by only one or the other

holoenzyme under other conditions. Even though a

promoter might be transcribed by both enzymes under

some conditions, changes in the environment that alter

template geometry (e.g. superhelicity) or solute concen-

trations (e.g. anions or cations) might be sufficient to alter

the ratio of transcription by the two holoenzymes

(Ishihama, 2000).

It has been reported that the two holoenzymes possess

different tolerances for high concentrations of K-glutamate

or trehalose, and that this favours specific promoter

recognition by EsS (Ding et al., 1995; Kusano and

Ishihama, 1997; Nguyen and Burgess, 1997). We

compared the activities of EsS and Es70 on the bolA1,

210 con, 235 con, and full con promoters in buffers

containing 100–800 mM K-glutamate or 10–400 mM KCl

(data not shown). As predicted from previous studies

(Leirmo et al., 1987), the anion glutamate was less

disruptive to RNAP–promoter interactions than chloride,

so transcription by both holoenzymes was generally higher

at the same cation concentration in glutamate buffers.

Tolerance for high salt concentrations may contribute to

the ability of certain promoters to be transcribed only by

EsS (Ding et al., 1995; Kusano and Ishihama, 1997).

However, the in vitro selected promoters described here

are transcribed by both holoenzymes at high osmolarity;

high salt does not result in holoenzyme specificity.

The degree of negative supercoiling of the bacterial

chromosome, on average, is somewhat lower in stationary

phase than in exponential growth, and it has been

suggested that reduced supercoiling may enhance

transcription by EsS rather than by Es70 (Kusano et al.,

1996). Our promoter selections were performed on DNA

fragments (linking number¼ 0), but the promoters were

also transcribed efficiently by both holoenzymes on

supercoiled plasmid templates (linking

number¼<20.06). Therefore, it is probable that the

Promoter recognition and discrimination by Ess RNAP 949

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

same consensus hexamers are recognized best by both

EsS and Es70 on templates with a wide range of

superhelicities. Nevertheless, as with changes in osmo-

larity, conditions that alter template geometry could

potentially favour transcription by one holoenzyme or the

other in a specific promoter context.

Conclusions

We have shown for the first time that two RNAP

holoenzymes present in the same organism recognize

the same consensus hexamer sequences. This poses a

potential problem for promoter specificity, but we have

proposed a model that might resolve this dilemma. The

principle of tolerance for different deviations from the same

consensus sequence was recognized long ago as the

basis for differential binding of the Cro and cI repressor

proteins from bacteriophages l and 434 (e.g. Hochschild

et al., 1986; Harrison and Aggarwal, 1990; Albright and

Matthews, 1998). Cro and cI bind to the same consensus

operator sequences, but the two proteins prefer different

non-consensus bases at particular positions in the DNA

sequence.

Differential tolerance of two holoenzymes for certain

deviations from the same consensus is apparently not the

method used for discrimination between the other E. coli

RNAP holoenzymes, as the other E. coli s factors have

diverged from each other more than sS and s70 and

recognize qualitatively different DNA sequences. Even in

the case of promoters recognized by EsS versus Es70,

context is important to the effects of individual promoter

positions on RNAP recognition, and thus it is difficult to

predict which deviations from consensus account for

discrimination simply by inspection of a particular promoter

sequence. However, we suggest that the concept of

tolerance for different deviations from the same consensus

sequence as a mechanism for achieving specificity should

be considered in transcription studies of organisms with

multiple closely related RNAP holoenzymes, a common

feature in bacteria. For example, recent genome analysis

suggests that Streptomyces coelicolor might have as

many as 40 ECF s factors (http://www.sanger.ac.uk/

Projects/S_coelicolor/ ). We speculate that the principle

described here for discrimination between E. coli EsS and

Es70 might be utilized for discrimination between members

of s factor families in other bacteria.

Table 1. Bacterial strains and plasmids.

Name Genotypea Source

StrainsRLG3499 VH1000¼MG1655 lacZ, lacI, pyrE 1 Gaal et al. (1997)UM122 rpoS::Tn10 Xu and Johnson (1995)RLG3773 VH1000 lI bolA1– lacZ(254 to 116) This workRLG3774 VH1000 lI bolA1– lacZ rpoS::Tn10 This workRLG3760 VH1000 lII bolA1– lacZ (254 to 116) This workRLG3764 VH1000 lII bolA1– lacZ rpoS::Tn10 This workRLG3750 VH1000 lI 210 con– lacZ (254 to 116) This workRLG3769 VH1000 lI 210 con– lacZ rpoS::Tn10 This workRLG3752 VH1000 lI 235 con– lacZ (254 to 116) This workRLG3770 VH1000 lI 235 con– lacZ rpoS::Tn10 This workRLG3754 VH1000 lI full con– lacZ (254 to 116) This workRLG3771 VH1000 lI full con– lacZ rpoS::Tn10 This workRLG5852 VH1000 lII CC 235 con– lacZ (240 to 12) This workRLG5857 VH1000 lII CC 235 con– lacZ rpoS::Tn10 This workPlasmidspRLG770 pKM2 with rrnB T1T2 terminators Ross et al. (1990)pRLG3405 pRLG770 with bolA1 promoter (–54 to 116) This workpRLG3747 pRLG770 with 210 con promoter (–54 to 116) This workpRLG3748 pRLG770 with 235 con promoter (–54 to 116) This workpRLG3749 pRLG770 with full con promoter (–54 to 116) This workpRLG3790 pRLG770 with AT 235 con promoter (–54 to 116) This workpRLG3791 pRLG770 with CT 235 con promoter (–54 to 116) This workpRLG3792 pRLG770 with GT 235 con promoter (–54 to 116) This workpRLG3793 pRLG770 with TA 235 con promoter (–54 to 116) This workpRLG3794 pRLG770 with TC 235 con promoter (–54 to 116) This workpRLG3795 pRLG770 with TG 235 con promoter (–54 to 116) This workpRLG3798 pRLG770 with AA 235 con promoter (–54 to 116) This workpRLG3796 pRLG770 with CC 235 con promoter (–54 to 116) This workpRLG3799 pRLG770 with GG 235 con promoter (–54 to 116) This workpRLG5881 pRLG770 with CC 235 con promoter (–40 to 12) This work

a. lI refers to lacZ fusion system I, and lII refers to lacZ fusion system II (see Experimental procedures ).Numbers in parentheses refer to sequence endpoints of DNA fragments used to construct promoter– lacZ fusions and plasmids.

950 T. Gaal et al.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

Experimental procedures

Promoter fragment library

In vitro selections with EsS were performed with libraries of86 bp promoter fragments containing 20 bp of random DNA

sequence. Non-template strand oligonucleotides (Integrated

DNA Technologies) for the 210 region selection contained(from 50 to 30) an Eco R1 site, bolA1 promoter sequences from

254 to 219, random sequences from 218 to 12 generatedfrom equimolar mixtures of the four nucleotides, bolA1

sequences from 13 to 116, and a HindIII site. Non-templatestrand oligonucleotides for the 235 region selection contained

an Eco R1 site, the ‘SUB’ sequence (Rao et al., 1994) from254 to 239, random sequences from 238 to 219, selected

210 region sequence #1 (Fig. 2B) from 218 to 12, bolA1sequences from 13 to 116 and a Hind III site. The template

strand for each of the two selections was generated byannealing 60 pmol of the non-template strand with 60 pmol of

a 21-mer complementary to the sequence from 13 to the HindIII site. The reactions were incubated in 25ml Sequenase

buffer (US Biochemical) for 5 min at 958C, slowly cooled toroom temperature, dNTPs were added to 1 mM, and the

annealed oligos were extended with 20 units of Sequenase for20 min at 378C. The DNAs were digested with Eco RI and

HindIII, and aliquots were cloned into M13mp18 for sequenceanalysis before selection with EsS to ensure that the

randomized regions contained approximately equal percen-tages of all four bases. The rest of the DNA was labelled at

both ends with [a232P]-dATP using Sequenase before use inthe selection.

RNA polymerases

EsS was prepared from core RNAP and highly purified sS

(Nguyen et al., 1993). The preparations contained no

detectable other s factors as judged on silver-stained gels(data not shown). EsS was reconstituted by mixing purified

core with a fivefold excess of sS (1mM) for 45 min at 308C.Es70 was reconstituted using the same core RNAP

preparation and a fivefold excess of purified s70. Increasingthe concentration of either s factor further did not increase

transcription (data not shown). Therefore, the two holoen-zyme preparations contain the same number of active RNAPs

(unless there are s molecules that bind to core but are notactive in transcription).

In vitro selection

The selection consisted of repeated cycles of RNAP binding,separation of the bound population from free DNA by

electrophoresis on 4% polyacrylamide gels in 0.5X Tris-Borate-EDTA buffer for 2–3 h at ,10 V cm21 and PCR

amplification (Fig. 1). For the initial 50ml binding reaction, 2mg(0.7mM) of the double-stranded DNA fragment library was

incubated with 20 nM EsS in 50 mM HEPES (pH 7.0), 100 mMKCl, 10 mM Mg-acetate, 0.1 mM dithiothreitol (DTT),

100mg ml21 BSA, 5% glycerol at room temperature for20 min. Then, 25mg ml21 heparin was added to prevent

further RNAP binding, and the samples were immediately

loaded on the gel. The RNAP–promoter complexes (0.5–5%

of the total DNA) were visualized by phosphorimaging,excised, eluted, extracted with phenol and precipitated with

ethanol. The recovered DNA was used as a template for PCRamplification using Taq DNA polymerase for 15 cycles (958C

for 1 min; 558C for 1 min; 728C for 1 min). For the 210selection, the PCR primers were complementary to 20 nt at

each end of the promoter DNA fragment, whereas in the 235selection, the primers were complementary to all except the

randomized positions (238 to 219). Aliquots were checkedby gel electrophoresis on 4% agarose gels and staining with

ethidium bromide for successful amplification. The amplifiedDNA was digested with HindIII, end-labelled and used for the

next cycle of RNAP binding. The binding time wasprogressively shortened, and the RNAP concentration was

gradually decreased, in subsequent selection cycles, until inthe last round the reaction time was 2 min and the RNAP

concentration was 4 nM. The progress of the selection wasfollowed after 10, 14 and 17 cycles by cloning samples into

M13 and sequencing a number of individual clones from the

selected population. The selection was stopped after 17cycles because inspection indicated that an obvious

consensus sequence had been reached.Heteroduplexes (which bind RNAP very efficiently) some-

times arose in the final cycles of the PCR from annealing of

single-stranded DNAs that contained different sequences inthe randomized region or from annealing of insertions and

deletions that arose from mistakes during oligonucleotidesynthesis or amplification. These appear on agarose gels as a

blurred region of stained material, migrating slower than theband of the expected size. If contamination from hetero-

duplexes was detected, 5ml of the reaction was used astemplate for an additional two cycles of PCR amplification in a

50ml reaction.

Strains and plasmids

Plasmids and strains are listed in Table 1. Cloning with M13and plasmids was carried out using standard techniques.

Plasmid templates for in vitro transcription were constructedby insertion of promoter fragments into pRLG770 (Ross et al.,

1990). Site-directed promoter mutations in 235 con weregenerated by PCR using plasmid pRLG3748 as template and

primers containing the desired sequence alterations. DNAsequencing was performed using a Sequenase kit supplied by

US Biochemicals.l lysogens were constructed containing promoter– lacZ

fusions as described (Rao et al., 1994). Two promoter– lacZ

fusion systems were used; ‘System II’ (Rao et al., 1994) wasused for measuring transcription from the bolA1 and CC235

con promoters as indicated. This fusion system has a very lowbackground, but it cannot tolerate very strong promoters.

‘System I’ (Rao et al., 1994), which has higher background butcan accommodate strong promoters, was used in all other

cases. The same promoter makes at least 6.7-fold more b-galactosidase in System II than in System I. The CC235 con

and bolA1 promoters in the lacZ fusions used in Fig. 7(Table 1; RLG5852, RLG5857, RLG5861 and RLG5862)

contain sequences only from 240 to 12 to eliminateinterference from the weak Es70 binding site upstream of

the 235 hexamer (see Fig. 4 and Nguyen et al., 1993). As the

reporter systems and the downstream endpoints in the

Promoter recognition and discrimination by Ess RNAP 951

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 939–954

promoters used for these fusions (12) are different from thoseused for the fusions in Fig. 5 (116), probably resulting in

different mRNA half-lives for the resulting transcripts, the b-galactosidase activities shown in Fig. 7 should not be

compared directly with those shown in Fig. 5. Strains lackingsS were constructed by transduction of rpoS::Tn10 from

RLG3237 (¼UM122; Xu and Johnson, 1995) with P1vir.

Measurement of promoter activity in vivo

Cells were grown in Luria–Bertani (LB) medium at 308C, and

b-galactosidase activity was measured at regular intervalsthroughout a growth cycle. To estimate promoter activity in

exponential phase, cells were grown to an A600 of < 0.25–0.5and then diluted 100-fold and grown again to ensure that b-

galactosidase, accumulated previously during stationaryphase, was minimized. The promoter activity at A600 0.25

and 0.5 was essentially identical. To estimate promoteractivity in stationary phase, b-galactosidase activity was

measured from cells grown to an A600 of 2.6 and 4.0 (in whichb-galactosidase activity was essentially identical).

Interference footprinting

Interference footprinting was performed as described in detail

elsewhere (Ross et al., 2001). The DNA contained, onaverage, one modified base (U instead of T; Devchand et al.,

1993; or A* instead of A; Min et al., 1996) per fragment,incorporated by PCR. In brief, RNAP (2 nM EsS or Es70) was

incubated with an excess of 32P end-labelled DNA fragment(10 nM) for 10 min at 228C in 50ml of 10 mM Tris-Cl (pH 7.9),

100 mM KCl, 10 mM MgCl2, and 1 mM DTT, so that about 5%of the DNA formed complexes. Heparin (final concentration

25mg ml21) was added, RNAP–promoter complexes wereseparated from unbound DNA and the DNA was eluted from

the gel. DNA fragments were cleaved at the position of A*incorporation by treatment with 1 M piperidine at 908C for

30 min. Fragments containing U were first treated with uracil

DNA glycosylase (UDG; NE Biolabs) and then cleaved withpiperidine. Fragments were analysed by phosphorimaging

(Molecular Dynamics). After electrophoresis on 10% denatur-ing polyacrylamide gels, lanes were normalized to correct for

loading differences and graphed using SigmaPlot (JandelScientific).

In vitro transcription

Reactions contained supercoiled plasmid DNA (20 ng), 10 mM

Tris-Cl (pH 7.9), 10 mM MgCl2, 1 mM DTT, 100mg ml21 ofBSA, 200mM ATP, GTP, and CTP, 10mM UTP, 4mCi [a-32P]-

UTP (NEN) and 10–800 mM KCl or K-glutamate. Transcrip-tion was initiated by addition of RNAP (1–4 nM EsS or Es70)

and terminated by addition of stop solution (Ross et al., 1993)after 15 min at 228C. Samples were electrophoresed on 5.5%

polyacrylamide 7 M urea gels and quantified byphosphorimaging.

Acknowledgements

We thank A. Ernst and G. Verdine (Harvard University) for

providing 7-deaza-7-nitro-adenine (A*), and M. Barker, J.Gralla, J. Helmann, A. Hochschild and R. Hengge-Aronis for

helpful discussions. This work was supported by NationalInstitutes of Health Grant GM37048 to R.L.G., by a Hatch

grant from the US Department of Agriculture to R.L.G. and byNIH grant GM28575 to R.R.B.

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