Introduction

Top Abstract Introduction Results Discussion Materials and methods References

The Escherichia coli RNA polymerase (RNAP) core enzyme (₂`) is capable of transcription elongation, but only the holoenzyme (<sub>2</sub>`) containing one of the seven  factors can carry out specific transcription initiation. Promoter recognition by the holoenzyme containing the major  factor (E⁷⁰) occurs through interactions of  with up to three promoter modules. The 10 hexamer (consensus sequence 5'-TATAAT-3') is recognized by  region 2.3-2.4 (Gross et al. 1998); the extended 10 region (consensus 5'-TGTGn-3') is recognized by  region 3.0 (Burr et al. 2000; Murakami et al. 2002b); and the 35 hexamer (consensus 5'-TTGACA-3') is recognized by  region 4.2 (Campbell et al. 2002). In addition, the C-terminal domains of the two  subunits (CTDs) are flexibly tethered to the  N-terminal domains (NTDs; Blatter et al. 1994) and at some promoters interact with specific sequences referred to as UP elements located upstream of the 35 hexamer (Ross et al. 1993, 1998; Gourse et al. 2000).

The UP element consensus sequence was determined by in vitro selection (full UP element consensus; Estrem et al. 1998). These results and other data suggested that UP elements can consist of one or two subsites, proximal and distal (Fig. 1). Consensus sequences for the proximal and distal subsites, each of which can interact with one of the two CTDs, were then identified individually by in vitro selection (Estrem et al. 1999). Extensive genetic analyses by random and alanine scanning mutagenesis identified seven amino acid side chains in CTD critical for DNA binding (Gaal et al. 1996; Murakami et al. 1996). These residues reside in two helix-hairpin-helix (HhH) motifs (Shao and Grishin 2000) that interact with UP element DNA in and across the minor groove (Ross et al. 2001; Yasuno et al. 2001). A high resolution X-ray structure of CTD bound to DNA confirmed the roles of the two HhH motifs of CTD in DNA recognition, and of five of the seven crucial side chains (R265, N268, G296, K298, S299) in direct or water-mediated DNA contacts (Benoff et al. 2002).

View larger version (30K): [in this window] [in a new window]   Figure 1.   DNA sequences from 27 to 59 (with respect to the transcription start site) for rrn P1 promoter constructs. The top group of promoters contains the rrnB P1 core promoter, and the bottom group contains the rrnD P1 core promoter. The sequences designated "4547" and "4549" contain the consensus proximal subsites derived by in vitro selection (Estrem et al. 1999). "rrnB P1 proximal" contains the rrnB P1 natural proximal subsite and an EcoRI linker upstream of position 50 (Rao et al. 1994) and was used in a previous study of effects of 70 region 4.2 mutants (Lonetto et al. 1998). "rrnB P1 full" contains the natural rrnB P1 full UP element. "Consensus full" contains the full UP element derived by in vitro selection (Estrem et al. 1998). "rrnB no UP" contains the "SUB" sequence fused to the rrnB P1 core promoter (Rao et al. 1994). "rrnD P1 full" contains the natural rrnD P1 full UP element. "rrnD no UP" is the rrnD P1 core promoter; sequence upstream of the EcoR1 linker is derived from pRLG770 (Ross et al. 1998; Table 1). The 35 hexamer is shown in lowercase and boldface. UP element sequences are in uppercase and boldface.

The CTD also plays a critical role in transcription activation by serving as a target of transcription factors [e.g., cyclic AMP receptor protein (CRP), Fis] that bind upstream of the core promoter region (for review, see Hochschild and Dove 1998; Busby and Ebright 1999), either adjacent to the 35 hexamer (Class II promoters; Savery et al. 1998; McLeod et al. 2002), or further upstream (Class I promoters; Aiyar et al. 2002; Savery et al. 2002). In addition to (or instead of) interacting with CTD, some activators binding at a Class II position can interact with ⁷⁰ region 4 (Kuldell and Hochschild 1994; Li et al. 1994; Kim et al. 1995; Lonetto et al. 1998; Landini and Busby 1999; Rhodius and Busby 2000; Nickels et al. 2002; Pande et al. 2002) or with the  N-terminal domain (NTD; Niu et al. 1996). In both Class I and II activation complexes, CTD can interact in a non-sequence-specific fashion with DNA adjacent to the activator binding site.

In addition to solution and X-ray structures of CTD and the CTD-DNA complex (Jeon et al. 1995, 1997; Wada et al. 2000; Benoff et al. 2002), structures are available of the RNAP holoenzymes from Thermus aquaticus (Murakami et al. 2002a) and Thermus thermophilus (Vassylyev et al. 2002), of the T. aquaticus holoenzyme bound to a short promoter fragment (Murakami et al. 2002b), and of T. aquaticus region 4 of  bound to a DNA fragment containing the 35 element (Campbell et al. 2002). These structures provide detailed information about many of the intersubunit interactions and RNAP-promoter interactions in the transcription initiation complex. However, because the flexibly tethered CTD is not resolved in any of the RNAP core or holoenzyme X-ray structures, no structural information is available concerning potential interactions of CTD with other RNAP subunits or with DNA in the context of an RNAP-promoter complex.

The location of the proximal UP element subsite, where CTD binds centered at approximately 42 (Newlands et al. 1991; Estrem et al. 1999; Ross et al. 2001), suggested that CTD might, like some activators at Class II promoters, interact with the region of ⁷⁰ bound to the 35 hexamer (region 4). Although a previous study using a promoter containing an rrnB P1 UP element did not support the model that CTD-⁷⁰ region 4 interactions are important for UP element function (Lonetto et al. 1998), we have reexamined this issue in the context of more recently identified UP element sequences consisting of the proximal subsites isolated by in vitro selection (Estrem et al. 1999). Hydroxyl radical protection and missing base interference footprinting studies suggested that these consensus proximal subsites were protected better by CTD when in an UP element lacking a distal subsite (Estrem et al. 1999; Ross et al. 2001; data not shown) and have larger stimulatory effects on transcription than the UP element tested previously (~170-fold compared with ~20-fold in vivo; Estrem et al. 1999).

Here, we report the identification of specific substitutions in ⁷⁰ region 4 and in CTD that reduce the function of certain UP elements in vivo and in vitro. We identify a position in promoter complexes containing these UP elements where accessibility to DNAse I cleavage is altered by the  and  mutants. This position is at the junction of the  and  binding sites. We construct a model for the CTD-DNA-⁷⁰ region 4 ternary complex based on our previous identification of the precise position where CTD interacts with the proximal subsite in the RNAP holoenzyme-promoter complex (Ross et al. 2001), on the X-ray structure of T. aquaticus  region 4 bound to DNA (Campbell et al. 2002), and on the x-ray structure of E. coli CTD bound to DNA (Benoff et al. 2002). Our genetic, biochemical, and modeling studies strongly suggest there is a functionally important interaction between specific surface-exposed residues in CTD and ⁷⁰. Together with data from the literature concerning residues in CTD required for transcription activation at Class I promoters, our work suggests that this interaction can contribute not only to the mechanism of UP element function, but also to activation by transcription factors.

	Results

Top Abstract Introduction Results Discussion Materials and methods References

Substitutions in ⁷⁰ region 4 reduce UP element function in vivo

We assessed the role of ⁷⁰ in UP element function by testing a previously described library of single alanine substitutions in region 4.2 (Lonetto et al. 1998). Strains were constructed in which a  prophage carried lacZ fused to either an rrnB P1 promoter containing a consensus proximal subsite (UP element 4547; Fig. 1) or an rrnB P1 core promoter lacking an UP element. [The UP element examined in a previous study (Lonetto et al. 1998), the rrnB P1 natural proximal subsite, is also shown in Fig. 1.] The host chromosome also contained rpoD fused to the trp promoter, resulting in control of wild-type ⁷⁰ expression by the trp repressor (Lonetto et al. 1998). These cells were then transformed with plasmids expressing mutant or wild-type ⁷⁰ (pGEX-2T ⁷⁰ and derivatives; see Materials and Methods).

-Galactosidase activities from the promoter-lacZ fusions were determined in the rpoD plasmid-containing strains when the chromosomally encoded ⁷⁰ was repressed (see Materials and Methods). Effects of 14 alanine substitutions in  on UP element function were determined by measuring the ratio of activities of promoters with and without UP elements and then calculating the fraction of the UP element effect in a strain expressing mutant  versus wild-type  (Fig. 2; see also Materials and Methods). One ⁷⁰ substitution, R603A, reduced the effect of the consensus proximal subsite by ~75%. Two substitutions, K593A and K597A, reduced the function of this UP element by ~25%-35%.

View larger version (63K): [in this window] [in a new window]   Figure 2.   Effects of 70 region 4 alanine substitutions on UP element function in vivo. -Galactosidase activities were determined from promoter-lacZ fusions in strains with rrnB P1 constructs containing either proximal subsite 4547 or the "SUB" sequence (no UP element) and with plasmids encoding either wild-type or mutant . Expression of the host rpoD gene was repressed (see Materials and Methods). The "UP element effect" in the presence of the plasmid-encoded mutant  was determined as the ratio of activities of promoters with or without the UP element, expressed as a fraction of the UP element effect in the presence of plasmid-encoded wild-type  (see Materials and Methods).

Two other ⁷⁰ mutants, E591A and R596A, had phenotypes not specific to UP element function. E591A reduced transcription from reporter fusions both containing and lacking UP elements by ~25% (data not shown). Although the corresponding residue in T. aquaticus ^A (E416) does not contact DNA directly in the crystal structure (Campbell et al. 2002), it has been proposed that this substitution alters  interactions with the 35 hexamer indirectly (Nickels et al. 2002). The effect of R596A on UP element function could not be evaluated in vivo, because this substitution led to induction of  prophages carrying the promoter-lacZ fusions and cell lysis. R596 has been implicated previously in activation of the P_RM promoter by cI (Li et al. 1994; Nickels et al. 2002). Therefore, the effect of this mutant  on UP element function was examined only in vitro (see following).

The effects of the  mutants on the function of a different UP element, the consensus full UP element (Estrem et al. 1998), were also measured. None of the  mutants reduced the function of this UP element by >20% (data not shown).

Substitutions in ⁷⁰ region 4 reduce UP element function in vitro

To determine whether the effects of  R603A, K593A, or K597A on proximal subsite function in vivo were direct, we examined transcription by RNAPs containing these mutant  subunits in vitro. Figure 3 shows the results from in vitro transcription experiments in which the same preparation of core RNAP was saturated with wild-type or mutant  subunits (see Materials and Methods for details). Similar results were obtained with RNAPs containing  subunits with histidine (his) tags or GST tags (data not shown).

View larger version (56K): [in this window] [in a new window]   Figure 3.   Effects of 70 region 4 alanine substitutions on UP element function in vitro. Promoters containing or lacking UP elements were transcribed in vitro with wild-type or  mutant RNAPs. (A) Transcript bands from representative gels. Templates contained either the rrnB P1 or rrnD P1 core promoter and the UP element indicated above the images of the transcripts (see Fig. 1 for sequences). RNAP holoenzymes were reconstituted from core RNAP and the purified wild-type or mutant  indicated at the left of each set of transcripts. Duplicate reactions are shown for most of the promoters. (B-E) Effects of  mutant RNAPs on UP element function. To calculate the "UP element effect" for each RNAP (mutant and wild type), we compared the amount of transcript from a promoter with an UP element to that from the same promoter lacking an UP element ("no UP"). The bars in the histograms represent the UP element effects obtained with the mutant holoenzyme divided by that with the wild-type holoenzyme. Error bars indicate standard deviations from at least three independent experiments. A ratio of 1.0 (horizontal line) indicates no difference in the UP element effect using the wild-type and mutant holoenzyme. (B)  RA603A RNAP. (C)  KA597A RNAP. (D)  KA593A RNAP. (E)  RA596A RNAP.

Transcription was measured from promoters containing or lacking UP elements (transcripts from representative gels are pictured in Fig. 3A; see Fig. 1 for promoter sequences). The UP element effects with the  mutant RNAPs were then calculated as ratios of the UP element effects with the wild-type RNAP (Fig. 3B-E).  R603A reduced the effects of each of two different consensus proximal subsites (UP elements 4549 and 4547) by ~50%-60% and of the natural rrnD P1 full UP element by almost 40%. In contrast, R603A did not reduce the effects of the natural rrnB P1 or consensus full UP elements (Fig. 3B). Thus, the results obtained in vitro were fully consistent with the results obtained in vivo, indicating that the effect of  region 4 on UP element function is direct.

The effects of  substitutions K597A (Fig. 3C) and K593A (Fig. 3D) on UP element function in vitro were smaller than the effects of R603A, reducing 4549 and 4547 UP element function by only ~15%-30%, and the effects of  K597A and  K593A on rrnD P1 UP element function were not different from the effect of wild-type  within error. Like  R603A,  K597A and  K593A did not decrease the effect of the consensus full or rrnB P1 full UP elements. Because we were not able to test the effect of  R596A in vivo (see earlier), we measured its effect on transcription in vitro.  R596A did not decrease UP element function in vitro (Fig. 3E).

In a few cases, effects of the rrnB full or consensus full UP elements were reproducibly enhanced by the  substitutions both in vitro (Fig. 3) and in vivo (data not shown). We will return to this subject in the Discussion.

Identification of CTD mutants causing defects in UP element proximal subsite function

The identification of  R603 as a residue important for the function of some (but not all) UP elements suggested that  region 4 might interact with CTD, but that this interaction might occur (or have functional consequences) only in some contexts. To identify residues in CTD that might interact with  region 4, we used our library of rpoA constructs coding for single alanine substitutions at each position in the CTD ( residues 255-329; Gaal et al. 1996; Kainz and Gourse 1998). We screened for mutants with a phenotype similar to that observed for the  mutants described earlier: a defect in consensus proximal subsite function and not in rrnB P1 full UP element function. Effects of a subset of the alanine library on rrnB P1 full UP element function were reported previously (residues 255-273 and 291-302; Gaal et al. 1996; Murakami et al. 1996). Effects of the remaining substitutions in CTD on full UP element function, as well as effects of the entire library on consensus proximal subsite function, have not been reported previously.

Plasmids carrying wild-type or mutant rpoA genes were transformed into strains containing one of three chromosomal promoter-lacZ fusions (rrnB P1 with the consensus proximal subsite, with the native rrnB P1 full UP element, or without an UP element). Effects of  substitutions on UP element function were determined by measuring the ratio of activities from promoters with and without UP elements and then determining the ratio of these UP element effects in strains expressing mutant versus wild-type  from the rpoA plasmids (Fig. 4; see also Materials and Methods).

View larger version (96K): [in this window] [in a new window]   Figure 4.   Effects of single alanine substitutions in CTD on UP element function in vivo. Strains contained plasmids encoding either wild-type or mutant rpoA alleles and rrnB P1 promoter-lacZ fusions containing an UP element [the consensus proximal subsite 4547 (A) or the native rrnB P1 full UP element (B)] or lacking an UP element. The "UP element effect" for each  mutant was determined as the ratio of -galactosidase activities from promoters with and without an UP element. The bars in the figure indicate the UP element effects with each plasmid-encoded  mutant as a fraction of the UP element effect with plasmid-encoded wild-type  (see Materials and Methods). Striped bars indicate a class of  mutants (D261A or E259A) reducing the effect of the consensus proximal subsite approximately twofold, but not reducing the effect of the full UP element. Dark bars indicate alanine substitutions that reduced the effects of both the consensus proximal subsite and the rrnB P1 full UP element. The effects of these substitutions on transcription are attributable to defects in UP element DNA binding (see text). Open bars indicate positions that are alanine residues in wild-type . Asterisks indicate positions where the plasmids contained either additional mutations or (for 312) wild-type rpoA (see Materials and Methods).

Twelve substitutions in plasmid-encoded  reduced the effect of consensus proximal subsite 4547 by at least 40% (D259A, E261A, T263A, R265A, N268A, C269A, L290A, L295A, G296A, K298A, S299A, and E302A; Fig. 4A), despite moderation of the effect of the mutant subunit by the presence of the wild-type rpoA gene on the host chromosome. Two of these substitutions, D259A and E261A, reduced consensus proximal subsite function in vivo by 50%-60% but had little if any effect on rrnB P1 full UP element function (i.e., <10% different from wild-type ; Fig. 4B) or on consensus full UP element function (data not shown).  E261A also reduced rrnD P1 and 4549 UP element function by about 40% (data not shown), whereas D259A had smaller effects on the function of these UP elements (15%-25%; data not shown). D259A and E261A therefore constitute a distinct phenotypic class whose effects on UP element function are similar to those of  R603A. In the CTD-UP element complex, D259 and E261 are surface exposed and relatively distant from the DNA (Ross et al. 2001; Benoff et al. 2002). Furthermore, it was shown previously that substitutions for E261 do not affect DNA binding (Tang et al. 1994; Savery et al. 1998). Therefore, these residues are candidates for direct interaction with ⁷⁰ region 4.

The other 10 substitutions reduced the effect not only of the consensus proximal subsite, but also of the rrnB full UP element (Fig. 4B). Effects of all 10 substitutions on DNA binding can be rationalized from the positions of these residues in the X-ray structure of the CTD-DNA complex (Benoff et al. 2002), and most have been reported previously to disrupt binding of  to UP element DNA in vitro (Gaal et al. 1996; Murakami et al. 1996).

Several other alanine substitutions in CTD (D258A, L260A, L262A, V264A, S266A, I303A, and V306A) reduced consensus proximal subsite function by ~30%, that is, by less than the substitutions listed earlier. Like  D259A and  E261A,  D258A reduced proximal subsite 4547 (but not full UP element) function in vivo. The other substitutions reduced both 4547 and full UP element function, and generally affected full UP element function more than proximal subsite function (Fig. 4). We suggest that these residues may have indirect effects on DNA binding that are most prominent in the context of full UP elements. L314A, a residue that is surface exposed but located far from either the DNA binding surface of CTD or from D259 and E261, also reduced full UP element function (by ~50%) and had a slight effect on proximal subsite function. Further characterization revealed that the plasmid encoding L314A contained an additional, previously undetected, mutation coding for an arginine substitution for L281. L281 is not surface exposed. We suspect that introduction of a large basic side chain at this position disrupts the (HhH)₂ fold sufficiently to alter the DNA binding surface indirectly.

E261A and D259A reduce UP element proximal subsite function in vitro

To determine whether E261A and D259A affect UP element function directly, we reconstituted RNAPs using purified wild-type ,  E261A, or  D259A, and transcription was measured in vitro from promoters containing or lacking UP elements (Fig. 5A). Consistent with the results obtained in vivo, E261A reduced the function of the consensus proximal subsites (4547 and 4549) by ~40% and of the rrnD P1 full UP element by ~30%, and had little or no effect on the function of the rrnB P1 full and consensus full UP elements (Fig. 5B). Although the effects of E261A were relatively small, they were very reproducible in multiple experiments and with different RNAP preparations. Although only a subset of these templates was tested with D259A RNAP, it had similar, albeit not identical, effects on UP element function as E261A RNAP (Fig. 5C). As in the in vivo experiments, D258A RNAP had smaller effects on proximal subsite function than either E261A or D259A RNAP (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 5.   Effects of  E261A and  D259A on UP element function in vitro. Promoters containing or lacking UP elements were transcribed in vitro with wild-type or  mutant RNAPs. (A) Transcript bands from representative gels. Templates contained either the rrnB P1 or rrnD P1 core promoter and the UP element indicated above the images of the transcripts (see Fig. 1 for sequences). RNAP holoenzymes were reconstituted from purified subunits using the wild-type or mutant  indicated at the left of each set of transcripts (Materials and Methods). Duplicate reactions are shown. (B-C) Effects of  mutant RNAPs on UP element function. To calculate the "UP element effect" for each RNAP (mutant and wild type), we compared the amount of transcript from a promoter with an UP element to that from the same promoter lacking an UP element ("no UP"). The bars in the histograms represent the UP element effects obtained with the mutant holoenzyme divided by the UP element effects obtained using the wild-type holoenzyme. Error bars indicate standard deviations from at least three independent experiments. A ratio of 1.0 (horizontal line) indicates no difference in UP element effect using the wild-type and mutant holoenzyme. (B)  E261A RNAP. (C)  D259A RNAP.

R603A decreases initial binding of RNAP (K_B)

Previous kinetic analyses indicated that the native rrnB P1 UP element increased the overall association rate (k_a) of RNAP with the rrnB P1 promoter ~20-30× in vitro (Rao et al. 1994). Most of this stimulation resulted from increasing the initial binding constant, K_B, but there also appeared to be an increase in the rate of isomerization to the open complex, k_f (Rao et al. 1994). Later studies showed that an UP element sequence fused to a synthetic promoter could facilitate isomerization (Strainic et al. 1998). We carried out kinetic studies to determine the step(s) responsible for the effect of  R603A on UP element function.

Concentrations of active wild-type and  R603A reconstituted RNAPs were determined, and the rates of formation of heparin-stable complexes on a promoter containing a consensus proximal subsite (UP element 4549) were measured as a function of active enzyme concentration using a filter binding assay (Roe et al. 1984; Rao et al. 1994; Barker et al. 2001). The composite association rate constant, k_a, has contributions from both initial binding (K_B) and subsequent isomerization (k_f) steps (k_a = K_Bk_f). k_a and k_f were determined from both linear and nonlinear analysis of k_obs as a function of RNAP concentration (see Materials and Methods). A linear representation of the data [Tau plot; 1/k_obs vs. 1/(RNAP); McClure 1980] is shown in Figure 6.

View larger version (17K): [in this window] [in a new window]   Figure 6.   Effects of  R603A on association of RNAP with the rrnB P1 promoter containing the consensus proximal subsite 4549. Association rate constants, kobs, were determined at different RNAP concentrations, and the results are shown as a Tau plot (McClure 1980). The kinetic parameters, ka = KBkf (determined from the nonlinear fit described in Materials and Methods), indicated that  R603A reduced ka, the observed second order association rate constant, by eightfold, all of which was caused by a decrease in the initial binding constant, KB. Kinetic parameters: ka (wild-type RNAP) = 9.7 ± 0.8 × 106 (M1sec1); ka (R603A RNAP) = 1.2 ± 0.1 × 106 (M1sec1); kf (wild-type RNAP) = 1.3 ± 0.1 × 102 (sec1); kf (R603A RNAP) = 2.3 ± 0.6 × 102 (sec1).

The value of k_a for  R603A RNAP was eightfold smaller than for wild-type RNAP under these solution conditions, even though isomerization was slightly faster (Fig. 6 legend). As a consequence, the equilibrium binding constant K_B (K_B = k_a/k_f) for  R603 RNAP was 14-fold smaller than for wild-type RNAP. Kinetic parameters determined for RNAPs reconstituted with  E261A versus wild-type  indicated that  E261A reduced the association rate 2.7-fold, and, as with  R603A RNAP, the reduction resulted largely from a change in K_B (data not shown). The complexes formed with either wild-type or  R603A polymerase showed no dissociation over the time course of the experiments (data not shown). The combination of these association and dissociation kinetic data demonstrate that the  region 4-CTD interaction primarily affects recruitment of RNAP to the promoter, rather than subsequent steps in the transcription mechanism. The larger defect caused by  R603A in Figure 6 versus Figure 3 could derive either from the greater sensitivity of the kinetic assay or from an UP element-independent component contributing to the effect of the mutant. Studies addressing this issue are in progress.

R603A and  E261A alter the structure of the transcription initiation complex

We asked whether  E261A or  R603A affected the structure of the RNAP-promoter complex using DNAse I footprinting. A selection of these footprints is shown in Figure 7A-D. On the 4547 promoter (a template where these mutants reduced UP element function both in vitro and in vivo), the footprints with  E261A and wild-type RNAP were virtually identical, except at position 38 on the template strand (numbering refers to the phosphodiester bond connecting 38 and 39). This position, which is located at the junction of the proximal subsite and the 35 element, was more accessible to DNAse I in the  E261A RNAP complex (Fig. 7A) than in the wild-type RNAP complex (Fig. 7A, inset). Likewise, on the 4549 promoter (another template where these mutants reduced UP element function both in vitro and in vivo), template strand position 38 was more accessible to DNAse I in the  R603A (Fig. 7B, see inset) or  E261A (data not shown) RNAP complex than in the wild-type RNAP complex. In contrast, cleavage at 38 was not altered in promoter complexes containing the mutant RNAPs and the rrnB P1 full UP element ( R603A RNAP, Fig. 7C;  E261A RNAP, data not shown), where these mutants did not affect UP element-dependent transcription. Similarly, altered cleavage by DNAse I was not observed with  R603A versus wild-type RNAP on the lacUV5 promoter (which does not contain an UP element; Ross et al. 1998; Fig. 7D). These results suggest that the RNAP substitutions alter the structure at the junction of the binding sites for CTD and  region 4, but only at promoters where the mutant RNAPs affect UP element-dependent transcription (see also Discussion).

View larger version (57K): [in this window] [in a new window]   Figure 7.   DNAse I footprints of promoters in the presence of wild-type and mutant RNAPs. Footprints were performed on rrnB P1 containing UP element 4547 (A), UP element 4549 (B), the rrnB P1 full UP element (C), or the lacUV5 promoter (D; see Materials and Methods). The regions in DNA protected from DNAse I cleavage and the regions corresponding to the UP elements are indicated under the gel images. The RNAP used in each lane is indicated at the left of the gel images: wild-type RNAP (WT), E261A RNAP (E261A),  R603A RNAP (R603A), or no RNAP (). Superimposed scans of gel lanes are shown above the images. Scans corresponding to footprints with mutant RNAPs are red, footprints with wild-type RNAP are blue, and footprints without RNAP are gray. Red arrows indicate position 38, and this region is magnified in the inset in each panel. Footprints of an E261A RNAP-4549 promoter complex were very similar to those shown in A, and footprints on an E261A RNAP-rrnB P1 full UP element promoter complex were very similar to those shown in C (data not shown). On the 4547 (A) and 4549 (B) promoters, position 38 was more sensitive to DNAse I cleavage in the complexes formed with the mutant RNAPs than with the wild-type RNAP.

Structure-based identification of an interaction between CTD and  region 4

Our studies suggest that an interaction between  R603 and  D259 and/or E261 plays an important role in UP element-dependent transcription at certain promoters. High resolution X-ray structures of E. coli CTD bound to a DNA fragment containing an A-tract (Benoff et al. 2002) and of T. aquaticus ^A domain 4.2 bound to a DNA fragment containing a 35 hexamer (Campbell et al. 2002) indicate that these residues are surface exposed and relatively distant from the DNA. (T. aquaticus ^A region 4.2 is 79% similar or identical to E. coli ⁷⁰ region 4.2.) In order to determine whether  R603 and  D259 and/or E261 are likely to be in close proximity, we juxtaposed the structures of the CTD-DNA complex and the  region 4-DNA complex to model a ternary complex, positioning the center of the CTD binding site precisely 6 bp from the upstream end of the 35 hexamer (promoter position 42; sequence numbering from rrnB P1). This placement of CTD relative to the 35 element was based on detailed information provided by protection and interference footprinting studies of RNAP on rrnB P1 promoters containing consensus proximal subsites (UP elements 4547 and 4549; Ross et al. 2001).

Figure 8 indicates that when the CTD and  region 4 binding sites are thus aligned, residues D259 and E261 of CTD are in very close proximity to R429 of T. aquaticus ^A (which corresponds to R603 of ⁷⁰; ⁷⁰ residue numbers are used on the figure), in complete agreement with their proposed interaction. ⁷⁰ Residues K593 and K597 (corresponding to ^A K418 and K422, respectively), which had small effects on UP element-dependent transcription (Figs. 2, 3), are not in the proposed - interface and are discussed following. Position 38, whose accessibility to DNAse I was increased when the proposed functional interaction between  and  was altered by mutation, is on the DNA surface opposite to the binding sites of  and . This suggests that the functional interaction between  and  distorts the DNA, altering the width of the minor groove, and thereby reducing access to cleavage by DNAse I (see Discussion).

View larger version (83K): [in this window] [in a new window]   Figure 8.   Structure-based model for the interaction between CTD and  region 4. The structure of the E. coli CTD-DNA complex is from Benoff et al. (2002), and the structure of the T. aquaticus A region 4.2 to 35 region DNA complex is from Campbell et al. (2002). CTD is shown centered at 42, 6 bp upstream of the upstream boundary of the 35 hexamer (position 36; numbering from rrnB P1), based on results from extensive footprinting studies of a complex containing RNAP and rrnB P1 promoters with UP elements 4547 and 4549 (Ross et al. 2001). DNA from 49 through 38 is from the CTD-DNA structure, and DNA from position 37 through 28 is from the A region 4-DNA complex. CTD is shown in ribbon form in white, with  D259 and  E261 in spacefill in blue. T. aquaticus A region 4.2 is shown in ribbon form in green (labeled with corresponding E. coli amino acid numbers).  R603 (spacefill in red) corresponds to A R429, and  K593 and  K597 (spacefill in light pink) correspond to A K418 and K422, respectively. DNA is shown in stick form. The views in A and B differ by a 90° rotation. The models were constructed using Insight II. The phosphodiester linkages on both strands between positions 38 and 37, the junction of the CTD-DNA and A region 4.2-DNA structures, are not modeled. The two complexes are positioned to produce a continuous B-form DNA helix because the degree of DNA bending, if any, at their junction is not known. The DNAse I cleavage site on the template strand between 38 and 39 (referred to here as 38), which is affected by the proposed - interaction (Fig. 7 and text), is indicated by a magenta sphere. The upstream DNA in the structure of  bound to the 35 region (Campbell et al. 2002) is bent toward , and K593 interacts with nontemplate strand position 39 [38 in the promoter used by Campbell et al. (2002) corresponds to 39 in rrnB P1]. This DNA distortion is not modeled in our complex (because the upstream DNA comes from the CTD-DNA structure), and  K593 is closer to 38 than to 39.

	Discussion

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Interaction of CTD and ⁷⁰ region 4.2

The genetic and biochemical results presented here strongly suggest there is a functional interaction between acidic side chains D259 and E261 on CTD and basic side chain R603 on ⁷⁰. The proposed interaction is based on several criteria: (1) the identification of alanine substitutions at these positions, leading to defects in UP element function in vivo and in vitro at the same subset of promoters; (2) the identification of a structural alteration (detected by altered DNAse I sensitivity of promoter position 38) in complexes containing either  E261A RNAP or  R603A RNAP; (3) the proximity of  residues D259 and E261 to  residue R603 in a structure-based model of the ternary complex formed by CTD,  region 4, and DNA.

The structure-based model (Fig. 8) provides a clear explanation for our genetic and biochemical observations. However, we emphasize that the model is based on a complex containing region 4.2 from T. aquaticus ^A, not from E. coli ⁷⁰. Therefore, although the ⁷⁰ and ^A sequences are very similar, confirmation of the model awaits solution of a structure of a ternary complex with subunits derived from the same bacterium.

Although  D259,  E261, and  R603 are positioned appropriately to interact, our model of the ternary complex suggests that two other nearby residues in  that slightly affected UP element-dependent transcription (K593 and K597) are unlikely to interact directly with CTD. In the 35 hexamer-T. aquaticus ^A region 4 complex (Campbell et al. 2002), K418 (⁷⁰ K593) contacts the DNA backbone at the promoter position corresponding to 39 (see also Fig. 8 legend). K593 mutants also reduce activator-dependent transcription (e.g., Lonetto et al. 1998; Landini and Busby 1999; Nickels et al. 2002), suggesting that the same K593-DNA backbone interaction plays a role in both UP element function and in activation by transcription factors. T. aquaticus ^A residue K422 (⁷⁰ K597) does not contact DNA in the crystal structure (Campbell et al. 2002). Although the mechanism responsible for its small effect on UP element function therefore remains to be determined, one possibility is that it alters DNA binding indirectly by affecting K593. Our results do not exclude potential roles for other nearby residues in , although the in vivo data suggest that these effects are small, if they occur.

Our data suggest that CTD residues E261 and D259 contact  region 4.  D258 could also be part of the patch contacting  because the alanine substitution at this position slightly reduced proximal subsite 4547 (but not full UP element) function. However, in our model of the complex (Fig. 8),  D258 is not as close to the  interface as E261 and D259, suggesting that either the DNA is distorted in the actual complex, slightly changing the orientation of D258 with respect to , or that D258A affects UP element function indirectly by altering the E261 and/or D259 interaction with  R603. It is possible that  subunits containing E261A, D259A, and D258A would cause greater defects in UP element-dependent transcription than the single-substitution mutants.

Context dependence of the CTD- interaction

The proposed CTD- interaction affects use of three different UP elements tested here: two consensus proximal subsites, originally identified by in vitro selection (Estrem et al. 1999), and the native UP element from the rrnD P1 promoter. The proximal subsite in the rrnD P1 UP element more closely resembles the consensus proximal subsites than the proximal subsites in the other three promoter constructs tested (which were unaffected by the proposed CTD- interaction; rrnB P1 full, consensus full, or rrnB P1 proximal; Fig. 1). Although further studies will be required to understand the context dependence of the CTD- interaction, we propose two potential (nonexclusive) explanations:

(1) The context dependence of the CTD- interaction reflects differing contributions of the proximal subsite to overall UP element function. This model is suggested by results from interference footprinting experiments, in which missing bases in the proximal subsite greatly reduced RNAP binding to a promoter containing only the consensus proximal subsite, but did not affect binding to promoters containing the native rrnB P1 full UP element or the consensus full UP element (Ross et al. 2001; data not shown). Furthermore, the proximal subsite was less strongly protected from hydroxyl radical attack by CTD in promoter complexes containing both subsites than in complexes containing only the proximal subsite (Newlands et al. 1991; Estrem et al. 1999).

It is possible that, in some full UP elements, a reduced contribution of the proximal subsite to transcription results from a decrease in CTD occupancy of this subsite, thereby reducing effects of the proposed - interaction. The mechanism by which the distal subsite might reduce occupancy of the proximal subsite remains unclear, however. Perhaps the distal subsite in some UP elements binds both CTDs at closely adjacent positions along the minor groove, as observed in the crystal of the CTD-DNA complex (Benoff et al. 2002). This configuration would explain why the length of the protected region is greater for the distal subsite than for the proximal subsite in hydroxyl radical footprints and missing base interference footprints of full UP elements (Newlands et al. 1991; Estrem et al. 1998, 1999; Ross et al. 2001). We speculate that, at some promoters containing two UP element subsites, transcription might be favored by having both CTDs positioned at the distal subsite. The slight transcription enhancement observed at these promoters by mutations that inhibit - interactions might be explained by reduced competition for CTD at the distal subsite by CTD bound at the proximal subsite (see Results).

(2) The context dependence of the effect of the CTD- interaction could also reflect effects of local DNA sequence near the junction of the  and  binding sites. Slight differences in the mode of DNA binding might affect the spatial separation of the relevant side chains in  and . For example, differences in the proximal subsite DNA sequence could affect the trajectory of the DNA and thereby the orientation of the bound CTD in the promoter complex. Binding of  region 4.2 to the 35 hexamer induces a bend in the DNA of about 36° (Campbell et al. 2002), facilitating interactions between  and the DNA backbone on the nontemplate strand immediately upstream of the 35 hexamer. It is conceivable that differences in local DNA sequence could affect these -DNA backbone interactions, thereby affecting the orientation of  R603 at the - interface.

We note that the extent of DNAse I cleavage of template strand position 38 in wild-type RNAP-consensus proximal subsite promoter complexes differs from that in some other promoter complexes, where cleavage at this position is enhanced by binding of RNAP (Fig. 7C,D; Ross et al. 1993; Craig et al. 1995). We suggest that the major DNA bend proposed to occur at this position in many promoter complexes (Craig et al. 1995) is less pronounced when there is an interaction between CTD and ⁷⁰ region 4.

CTD- interactions at other promoters

It is likely that the CTD- interactions described here are important for many bacterial promoters. UP elements are present in promoters transcribing rRNAs, tRNAs, and mRNAs, and good proximal subsites are more frequent than good distal subsites or good full UP elements in E. coli promoters (Estrem et al. 1999). In this regard, it has been reported that substitutions for E261 reduce factor-independent transcription from the metE promoter in vitro and in vivo (Jafri et al. 1995, 1996).

The same CTD- interaction that facilitates UP element proximal subsite function apparently also plays a role in activation by transcription factors at Class I promoters, where CTD is at the same position relative to  as at promoters with proximal subsites (Busby and Ebright 1999).  E261A reduces Class I CRP-dependent activation of the lac promoter (Tang et al. 1994; Savery et al. 2002) and Class I TyrR-dependent activation of the mtr promoter (Yang et al. 1997). E. coli strains haploid for E261K also display a variety of other phenotypes (Jafri et al. 1995, 1996), suggesting that this surface on CTD plays a role in multiple cell functions.

Residues in ⁷⁰ region 4.2 have also been identified as activation determinants at several Class II promoters, for example, alkA (Landini and Busby 1999), melRcon (Lonetto et al. 1998), narG and dmsA (Lonetto et al. 1998), and P_RM (Nickels et al. 2002), where transcription factors bind just upstream of the 35 hexamer. Not surprisingly, different amino acid side chains in  may interact with different activator proteins at these promoters: R603 for CRP, FNR, and Ada (Lonetto et al. 1998; Landini and Busby 1999) and R588 for  cI (Nickels et al. 2002). Therefore, at Class II promoters,  region 4 interactions with activator proteins might function like -CTD interactions at the UP element-dependent promoters described here.

It was suggested previously that  R603A might cause a general transcription defect (Lonetto et al. 1998) because its effects on Class II CRP-dependent transcription did not depend on the putative  interaction surface on CRP, it affected transcription from all promoters tested, and strains expressing R603A displayed reduced growth rates in the defined media used. As indicated earlier, it is possible that R603A has general effects in addition to its specific effects on UP element-dependent transcription (Fig. 3), although it did not reduce the growth rate of our strain in the rich medium used (data not shown).

Amino acid sequences C-terminal to ⁷⁰ R599 diverge in the alternative  factors (Gruber and Bryant 1997). Future studies will be required to determine whether alternative - interactions can contribute to transcription by other holoenzymes.

In conclusion, our results indicate that a  region 4-CTD interaction plays a role in transcription from a subset of UP element-dependent promoters, where an CTD protomer binds to the DNA minor groove adjacent to the 35 hexamer, adjacent to  region 4.2 bound in the DNA major groove. We suggest that this same interaction participates in activation by transcription factors at Class I promoters, where an CTD protomer binds at a comparable position in the activation complex.

Ebright and colleagues (H. Chen and R. Ebright, unpubl.) independently have proposed that an interaction between the 261 determinant of CTD and region 4 of ⁷⁰ plays roles in activator-dependent and UP element-dependent transcription.

	Materials and methods

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Bacterial strains and plasmids

Strains and plasmids are listed in Table 1. Effects of rpoA and rpoD mutations on promoter activities were measured in strains carrying promoter-lacZ fusions on a single copy  prophage. Effects of rpoA mutations were measured in derivatives of NK5031 (Gaal et al. 1989), and effects of rpoD mutations were measured in derivatives of VH1000 (Gaal et al. 1997). The trp promoter-rpoD chromosomal fusion coding for wild-type ⁷⁰ was introduced into  lysogens by P1 transduction from CAG20153 (Lonetto et al. 1998