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J. Biol. Chem., Vol. 276, Issue 34, 31891-31896, August 24, 2001

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Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ⁷⁰ in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes^*

Mark Tomsic, Laura Tsujikawa, Gianina Panaghie, Yang Wang, Joseph Azok, and Pieter L. deHaseth^§

From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935

Received for publication, June 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amino acid residues in region 2 of ⁷⁰ have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the 10 region. We concluded that two basic amino acids (Lys⁴¹⁴ and Lys⁴¹⁸) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr⁴³⁰ and Trp⁴³³). It is likely that these four amino acids, which are close to each other in the structure of ⁷⁰, together are involved in the nucleation of the strand separation process.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RNA synthesis in prokaryotes is carried out by a multi-subunit RNA polymerase commonly referred to as the core enzyme (E).¹ For promoter recognition, a sigma (initiation) factor is required; it interacts with the core polymerase to yield the holoenzyme (E), which is able to form an initiation-competent complex at promoter sequences in a multistep process involving conformational changes in both the protein and the DNA (1-3). A striking feature of such a complex is a region of strand separation that spans about 14 base pairs from the upstream edge of the conserved 10 promoter element to just beyond the start site of transcription initiation (4). It is thought that, kinetically, strand separation initiates in the 10 region and proceeds in a downstream direction. Measurement of the size and location of the region of strand separation as a function of temperature shows that at low temperatures a small single-stranded region can be detected that, as the temperature is increased, expands toward the start site (3, 5-7). In addition, the introduction of nicks and mismatches in the 10 region is more effective in the acceleration of open complex formation than if such distortions are introduced at a more downstream position (8, 9).

The predominant sigma factor in Escherichia coli, which enables recognition of promoters of housekeeping genes, is referred to as ⁷⁰. Sequence comparison has shown that a large group of sigma factors shows significant homology to ⁷⁰. Four regions of sequence conservation have been identified, of which some have been subdivided to reflect the most extensive sequence conservation (10). A large body of data has implicated region 2.3 of the main sigma factors of E. coli, Bacillus subtilis, and other prokaryotes in the nucleation of the strand separation. This process eventually results in the formation of the active or open complex, possibly by facilitating base flipping of the highly conserved A at 11 of the nontemplate strand. The supporting experimental evidence has been derived from analysis of the effects of alanine substitution for aromatic amino acids on open complex formation (5, 11) and on the ability of RNA polymerase to interact with model substrates such as single-stranded DNA (12, 13) and duplexes carrying regions of unpaired DNA (14), also referred to as forked templates (15). The former are thought to model the unpaired regions of the strand-separated bubble, the latter the junction between double- and single-stranded DNA of the bubble. Based on the use of forked templates, Gralla and co-workers (14) have concluded that Tyr⁴³⁰ and Trp⁴³³ of region 2.3, which jut out of the body of the protein (16), are particularly important for the initiation of the strand separation process.

In this paper we demonstrate that alanine substitutions at the positions of Tyr⁴³⁰ and Trp⁴³³ lead to different effects on the interaction of E⁷⁰ with forked DNA. We provide new results in support of the idea that multiple aromatic amino acids jointly interact with single-stranded DNA downstream of the region where strand separation is initiated. Our results allow us to single out two basic amino acid residues in region 2 (lysines 414 and 418) as being particularly important for the interaction of RNA polymerase with DNA. Finally, we demonstrate that the substitutions of alanines for basic amino acids in region 2 have effects that are fundamentally different from those of substitutions for aromatic amino acids in the same region.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Oligonucleotides (oligos) were synthesized by Life Technologies, Inc. or Genset. Nonradioactive NTPs and dNTPs were purchased from Roche Molecular Biochemicals. CpA was from Sigma. [-³³P]ATP, [-³²P]ATP, and [-³²P]UTP were from PerkinElmer Life Sciences. DNA-modifying enzymes were purchased from either New England Biolabs or Roche Molecular Biochemicals. E⁷⁰ core enzyme was prepared as described (17) or purchased from Epicentre.

Methods

Deoxyoligonucleotide Labeling and Annealing-- 5' end-labeled DNA oligonucleotides were generated by incubation with [-³³P]- or [-³²P]ATP and T4 polynucleotide kinase (New England Biolabs) using established procedures. The two strands of forked DNA templates were re-annealed at concentrations of 100 nM (5' end-labeled nontemplate strand) and 150 nM (unlabeled template strand) in a buffer containing 25 mM Tris (pH 7.9 at 25 °C) and 50 mM NaCl by heating to 90 °C and slow cooling.

⁷⁰ Mutagenesis and Purification-- All manipulations of the ⁷⁰ coding region were performed on the pLHN12-His expression plasmid, an isopropyl-1-thio--D-galactopyranoside-inducible version of Pet11a vector from Novagen exactly as previously described (11). Site-directed single mutations were introduced using the QuickChange site-directed mutagenesis kit (Stratagene) and the appropriate primers according to the manufacturer's instructions. The mutagenized fragments were subcloned into the wild type pLHN12-His expression vector using the PstI and BamHI restriction enzymes, and the recombinant plasmids were resequenced (Molecular Biology core facility at Case Western Reserve University) to verify the lack of undesired mutations in the entire subcloned DNA. Successful clones were transformed into E. coli strain DH5a for maintenance and BL21(DE3) for over-expression.

Wild type and mutant ⁷⁰ bearing an amino-terminal (his)6 tag were purified from E. coli BL21(DE3) host expression cells containing pLHN12-His vectors with recombinant rpoD genes as described previously (11). For all experiments described here, RNA polymerase holoenzymes were reconstituted in storage buffer (10 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, 50% glycerol) from purified E. coli core enzyme (Epicentre) and purified ⁷⁰ in a molar ratio of 1:5 at 4 °C for 1 h. Proper interaction with core was verified by a gel mobility shift assay (11, 18).

Electrophoretic Mobility Shift Assays (EMSA)-- In experiments to determine the k_obs for heparin resistant (open) complex formation, we used a template in which the P_R promoter was inactivated by two mutations in its 35 region, whereas the P_RM promoter had an "up" mutation in its 10 region (TAGATT to TAGAAT). The experiments were carried out as described previously (11). Briefly, about 2-5 nM ³²P-labeled DNA fragments (obtained by polymerase chain reaction and polyacrylamide gel purification) were incubated with 200 nM E⁷⁰ at 37 or 20 °C in 20 ml HEPES buffer (30 mM HEPES, pH 7.5, 100 mM KCl, 1 mM dithiothreitol), containing 50 µg/ml bovine serum albumin, for various amounts of time. Each reaction was challenged with heparin to 100 µg/ml for 1 min prior to the addition of the loading solution and loading onto a 4% nondenaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide) run at room temperature. Frozen gels were autoradiographed and quantified by PhosphorImager (Molecular Dynamics). The radioactivity in each band, as the percentage of the total in the lane, was plotted versus the time of incubation with the E⁷⁰. The k_obs for the wild type and mutant RNA polymerases were determined by fitting the data to the equation y = Yf * (1  exp(t × k_obs)) + Yo, where y = the percent of open complexes formed, t = time of DNA/E⁷⁰ incubation, Yf and Yo are the limiting values for y, and k_obs is the pseudo-first order rate constant.

Interaction of E⁷⁰ with Single-stranded and Forked DNAs-- All reactions were performed in HEPES buffer (see above). Binding to single-stranded (ss) DNA was studied by incubating 5' ³²P-labeled DNA oligo (10 nM) and E⁷⁰ (65 nM) for 30 min at 25 °C followed by the addition of loading solution and loading onto a 5% nondenaturing gel, which was run at 4 °C (13). The intensity of bands corresponding to free and E⁷⁰-bound oligo was determined by PhosphorImager (Molecular Dynamics). Binding to forked templates was determined similarly, except that the reactions were subjected to a 10-min challenge with 100 µg/ml heparin prior to loading onto the gel at 25 °C (15). To determine the stability of E⁷⁰-forked DNA complexes, 40-µl solutions were prepared containing 10 nM ³²P-labeled forked DNA template and 65 nM E⁷⁰ in HEPES buffer. After a 30-min incubation at 25 °C, heparin was added to 100 µg/ml, and at regular time intervals 4.5-ml aliquots were removed for analysis on a 5% nondenaturing gel as described above.

DNase I Footprinting-- DNase I footprinting experiments were carried out as described (11) on DNA fragments with wt sequence, for which the observed footprint is at the P_R promoter. The DNA substrates were obtained by polymerase chain reaction using 5'-³³P labeled primers and purified on a 6% native polyacrylamide gel. RNA polymerase was incubated with labeled promoter DNA in 20 µl of HEPES buffer at 37 °C for 30 min. At the conclusion of the incubation period, heparin was added to a final concentration of 100 µg/ml. After the MgCl₂ concentration was adjusted to 10 mM the DNA was cut with 0.4 units of DNase I (Ambion) for 30 s at 37 °C. The reactions were terminated and analyzed on denaturing gels as described previously (11).

Abortive Initiation-- Abortive initiation assays were done essentially as described (19). For analysis of open complex formation at the P_R promoter, the unlabeled DNA fragment (2-5 nM) (obtained by polymerase chain reaction) and E⁷⁰ (200 nM) were incubated in transcription buffer at 37 °C for 30 min. After the incubation period heparin was added to a final concentration of 100 µg/ml, and reactions were further incubated for 5 min. To initiate transcription, CpA and ³²P-labeled UTP were added (providing final concentrations of 0.5 mM CpA, 50 µM UTP, and 10 µCi of [-³²P]UTP). The reactions were incubated for 15 min at 37 °C after which 5 ml of transcription stop solution (7 M urea, 0.1 M EDTA, 0.4% (w/v) SDS, 40 mM Tris-HCl (pH 8.0), 0.5% bromphenol blue, and 0.5% xylene cyanol FF) was added. The CpApU was separated from the substrates on a 20% denaturing polyacrylamide gel. Gels were frozen and exposed to film for 2-3 h. The intensities of the bands corresponding to UTP and CpApU were determined by densitometry.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ⁷⁰ mutant set used in this study is shown in Fig. 1a. It consists of alanine substitutions for basic and aromatic amino acids and one nonpolar residue (I435A; also I435L). All basic amino acids in region 2.3 as well as two residues in regions 2.2 and one in 2.4 are included. The aromatic amino acids (described in Ref. 11) are all in region 2.3; only the buried residue Phe⁴¹⁹ and residue Tyr⁴²¹ from this region were not substituted. Studies from Gralla and co-workers (14) have made use of an overlapping set of substitutions extending from Tyr⁴²⁵ in the C-terminal direction through Ile⁴⁵². The results presented here extend those of the latter two studies.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Sequences of the 70 variants and the DNAs used in this study. a, the sites of single alanine substitutions in 70 are indicated. b, ssDNA oligos. Both oligos have the sequence of the non-template strand of the PR' promoter, except in the 10 sequences (underlined), where the consensus oligo has the TATAAT sequence, whereas the 11C oligo has CATAAT. c, forked templates. The sequences are based on that of the PR' promoter of bacteriophage . The positions of the 10 and 35 elements are indicated by boxes. The short fork has just the overhanging A at position 11; the long fork has an entire consensus 10 sequence in the overhang.

With the exception of ⁷⁰ with alanine or leucine substitutions for Ile⁴³⁵, all were able to bind core as determined by a gel mobility shift assay (data not shown), which was carried out exactly as described (11). The Ile⁴³⁵ residue is highly conserved, probably for structural reasons, as it is buried in the ⁷⁰ structure. Even a conservative substitution of this residue with leucine apparently leads to major structural defects that interfere with ⁷⁰ core interactions. DNase I footprinting of complexes of the E⁷⁰ holoenzyme, reconstituted with wt ⁷⁰ or ⁷⁰ containing substitutions in basic amino acids of region 2 (see Fig. 1), and the P_R promoter is shown in Fig. 2. The E⁷⁰ and the promoter were incubated for 30 min prior to a heparin challenge for 1 min and exposure to DNase I for 30 s. With the exception of E⁷⁰ reconstituted with K414A ⁷⁰, all holoenzymes afforded complete protection over a region of DNA between 50 and +20. At 37 °C, for the P_R promoter, such an extended footprint is characteristic of the open RNA polymerase-promoter complex. Holoenzyme containing the Lys^{414 70} afforded only partial protection, consistent with the observation that this mutant E⁷⁰ forms open complexes with only about 25% of the DNA even after long incubation times, as compared with over 60% for the others (data not shown). No evidence was obtained for a heparin-sensitive complex with a "short" footprint, characteristic of a closed complex, with any of the E⁷⁰ tested bearing alanine substitutions for basic amino acids.

View larger version (64K): [in this window] [in a new window]   Fig. 2.   DNase I footprinting analysis of E70-DNA complexes formed using holoenzymes reconstituted with wild type and mutant 70. The DNA was labeled with 33P at the 5' end of the template strand. The outer lanes contained no added E70 (No RNAP, no RNA polymerase); the nature of the sigma factor used is indicated above the other lanes.

In Fig. 3, the rate of open complex formation at P_RM 10up for E⁷⁰ containing substitutions in basic amino acids is compared with the rates previously reported for E⁷⁰-bearing substitutions in aromatic amino acids (data from Ref. 11; note that YW bears alanine substitutions at positions 430 and 433; YYW, at 425, 430, and 433; FYW at 427, 430, and 433; and FYWW, at 427, 430, 433, and 434). Just as with the aromatic amino acids (5, 20, 21), the E⁷⁰ bearing Ala substitutions at basic amino acid residues Arg⁴²², Arg⁴²³, Lys⁴²⁶, and Arg⁴³⁶ confer cold sensitivity compared with E⁷⁰ reconstituted with wt ⁷⁰. However, at 37 °C these same four substitutions have relatively minor effects on E⁷⁰ function. In contrast, substitutions at Lys⁴¹⁴ and Lys⁴¹⁸ lead, even at 37 °C, to very slow formation of open complexes. The W433A substitution has an equally large effect. Only the E⁷⁰ reconstituted with triply or quadruply substituted ⁷⁰ had lower rates of formation of open complexes (this pattern is confirmed by abortive initiation data shown in Fig. 4; see below). Because of the low precision in the data for K414A, we were unable to assess whether the temperature dependence data obtained for this substitution were reliable.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Kinetics of open complex formation by mutant and wt E70. The promoter used was a PRM "up" promoter in the context of an inactivated PR (see Ref. 11). YW bears alanine substitutions at positions 430 and 433; YYW, at 425, 430, and 433; FYW at 427, 430, and 433; FYWW, at 427, 430, 433, and 434. The concentration of E70 was 200 nM. Open complex formation of heparin-challenged reaction mixes and kobs was assayed by EMSA, calculated as described under "Materials and Methods." All numbers are averages of at least two determinations. The values have been normalized to the wt values of 2.2 ± 0.6 min1 and 3.6 ± 0.2 min1 determined at 20 and 37 °C, respectively. Light bars, 20 °C; dark bars, 37 °C.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Open complexes formed with E70 containing mutant 70 are able to catalyze formation of a phosphodiester bond. DNA containing the wt PR promoter of bacteriophage  was incubated with wt and variant E70 for 30 min, whereupon the complexes were challenged with heparin for 1 min followed by addition of CpA and UTP and further incubation for 15 min prior to loading on gel to separate radiolabeled CpApU and UTP. The amount of label in the UTP and CpApU was quantified and the data normalized to the fraction of label in the CpApU product observed with wt E70.

Analogous results were obtained in experiments in which the formation of E⁷⁰-promoter complexes, competent for formation of the first phosphodiester bond, was monitored by determination of the synthesis of the trinucleotide CpApU from CpA and UTP. In these experiments, E⁷⁰ and promoter DNA are incubated for 30 min prior to a 1-min heparin challenge and addition of the RNA synthesis substrates. The amount of trinucleotide synthesis reflects the formation, during the 30-min incubation period, of complexes that were competent to initiate RNA synthesis. The ranking of the mutant E⁷⁰ in this experiment roughly mirrors that established by their relative k_obs of open complex formation, with K414A and K418A each being about as detrimental as multiple substitutions for aromatic amino acid residues. However, in contrast to the data shown in Fig. 3, here the K418A substitution is significantly less damaging than K414A. The lack of observed effects of the R422A and R436A substitutions in this experiment stands in contrast to their effect on the rate of open complex formation (Fig. 3). This likely reflects the relatively long preincubation between E⁷⁰ and the DNA in the abortive initiation experiments, which permits detection of only severe effects.

To better pinpoint the nature of the defects in the substituted sigma factors that are responsible for their impaired function, we employed the model templates shown in Fig. 1, b and c. To assess the extent to which the substitutions might impede the ability of E⁷⁰ to interact with the single-stranded DNA, and thereby render it less competent in propagating strand separation during open complex formation, we determined the ability of the mutant E⁷⁰ to interact with ssDNA. It had been shown previously that E⁷⁰ holoenzyme, but not free ⁷⁰ or core enzyme, is able to sequence-specifically interact with single-stranded DNA spanning the 10 promoter element and having the sequence of the non-template strand of promoter DNA (12, 13, 22). Two oligos were employed, bearing either the consensus TATAAT 10 sequence or a non-consensus variant, CATAAT (Fig. 1b). The results are shown in Fig. 5. Core, while not showing any sequence specificity, does have a relatively high affinity for both oligos in accordance with previous observations of its high affinity for ssDNA (23). Thus, the departure from such behavior, as observed with all of the reconstituted E⁷⁰ tested here, constitutes independent verification that the mutated ⁷⁰s indeed bind to core under the conditions of the experiment. All E⁷⁰ variants bind better to the consensus oligo, demonstrating that the assay was indeed detecting sequence-specific binding. The salient finding here is the poor binding that is observed with the E⁷⁰ bearing the Ala substitutions for positive amino acids in ⁷⁰ (K414A, K418A, R423A, and K426A), establishing an interaction of the DNA with amino acids residues on ⁷⁰ that extend beyond region 2.3 (Lys⁴¹⁴) and are contained on both the nearly parallel  helices 13 and 14. In addition, the Y425A substitution as well as triple and quadruple substitutions of alanine for aromatic amino acids are detrimental. The effect of the Y425A substitution (also seen for the equivalent amino acid in ^A (24)) is especially interesting as it is far removed on the structure from the location of residue Gln⁴³⁷, thought to interact with the T at position 12. It serves to define a potential path of the ssDNA on ⁷⁰ in an open complex.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Interaction of wt and mutant E70 with ssDNA. Complex formation was determined by EMSA as detailed under "Materials and Methods." The two oligos used differed in the sequence of the 10 region as shown in Fig. 1b. Light bars, consensus oligo; dark bars, 12C oligo.

Guo and Gralla (15) have established "forked" DNAs, modeling the junction between double- and single-stranded regions, as particularly useful model templates, able to form heparin-resistant complexes with E⁷⁰. We have employed here the "short fork" containing a minimal ss region consisting of just the A at 11, as well as the "long fork," where the ss extension covers the entire 10 sequence (see Fig. 1c). The results are shown in Fig. 6. E⁷⁰ containing ⁷⁰ with the Y430A and K418A single substitutions, as well as those containing multiple substitutions including Y430A, are particularly deficient in their ability to bind the short fork. On the other hand, the Y425A, R422A, and K426A substitutions, which drastically affect the ability of E⁷⁰ to bind ssDNA, have very small effects, if at all, as compared with the wt ⁷⁰. All E⁷⁰ variants bind the long fork more tightly than the short fork, in most cases so tightly that the ability to discriminate differences in binding affinities is likely to be outside the useful window of this experiment. However, with the long fork, the multiply substituted ⁷⁰ variants could be differentiated; the extent of binding decreases in the order YW > YYW > FYW > FYWW, indicating that substitutions at positions 425-434 affect the interaction of the long fork with E⁷⁰. The binding of the short fork may have reached the other extreme (background) with the E⁷⁰ containing the multiple substitutions. Thus it is not possible to conclude that the multiple substitutions do not affect the binding of the short fork and to infer from these data alone that, for the long fork, these substitutions mostly affect the interaction with the ssDNA tail downstream of 11A. However the data for the interaction of E⁷⁰ with single-stranded DNA (Fig. 5), where the binding affinity decreased in a similar order, YW > YYW > FYW  FYWW, would seem to lend support to such a conclusion.

View larger version (49K): [in this window] [in a new window]   Fig. 6.   The interaction of wt and mutant E70 with forked DNA. E70 was incubated with the forked DNAs as indicated ("Materials and Methods"); then the complexes were challenged with heparin for 1 min, after which the extent of complex formation was determined by EMSA. The sequences of the short (dark bars) and long (light bars) forks are shown in Fig. 1c.

We also determined the stabilities of the heparin-resistant complexes formed with E⁷⁰ containing the wt and mutant sigma factors. We show elsewhere² that formation of a stable complex between E⁷⁰ and the short fork DNA proceeds kinetically through a heparin-sensitive intermediate. At equilibrium, a substantial fraction of the heparin-sensitive complexes persist; upon addition of heparin these complexes dissociate with a rate that is too fast to measure by the manual mixing methods employed here.² Our experiments address the stability of the fraction of the complexes that dissociate with a slower rate. All substitutions that were assayed in the course of this study affect the stability of these complexes (see Fig. 7); the half-lives for the complexes formed with the variant E⁷⁰ are less than half of those for wt E⁷⁰, which is remarkably stable, with a half-life of over 100 min. The complex of Y430A E⁷⁰ with the short fork has a half-life of about 15 min, and substitutions of alanine for additional aromatic amino acids other than Tyr⁴³⁰, are not found to further destabilize the complex. The next most labile complexes were those formed with E⁷⁰ containing singly substituted ⁷⁰ K425A and F427A, which had half-lives of about 30 min and thus were twice as stable.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Stability of wt and variant E70-short fork complexes. After complexes had been allowed to form for 30 min, heparin was added, and aliquots were analyzed by EMSA for the extent of complex formation after various time intervals. The data obtained between 10 and 60 min of heparin addition were fit to a single exponential to determine the half-lives of the complexes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The experiments presented here were carried out to study nucleation of strand separation as orchestrated by region 2 of E. coli ⁷⁰. From the findings we conclude that both basic and aromatic residues are important for open complex formation: All basic residues examined, with the possible exception of Arg⁴²², play a role in open complex formation, as is most evident at 20 °C, but substitutions for Lys⁴¹⁴ and Lys⁴¹⁸ have the most prominent effects. Substitutions at Lys⁴¹⁴, Arg⁴²³, Lys⁴²⁶, and Arg⁴³⁶ affect ssDNA binding and at Lys⁴¹⁴ and Lys⁴¹⁸ formation of a heparin-resistant complex with forked DNA. We previously established the involvement of Tyr⁴²⁵, Phe⁴²⁷, Tyr⁴³⁰, and Trp⁴³³ in open complex formation. Here we show that Tyr⁴²⁵, Phe⁴²⁷, and Tyr⁴³⁰ are important for interaction with ssDNA, Phe⁴²⁷, Tyr⁴³⁰, and Trp⁴³³ for formation of a heparin-resistant complex with forked DNA and Tyr⁴³⁰ for its stabilization. Because Phe⁴²⁷ is buried (16), the effects of its substitution could be indirect. Trp³³⁴ may be involved in forked DNA binding (Ref. 14; see also Fig. 6) but we have insufficient data for this residue. Together with the data from Gralla's group (14), we conclude that residues of ⁷⁰ from 414 to 452, thus including helix 13 (16), the connecting loop, and helix 14, are involved in formation of an open complex.

We attempted to correlate the deficiency in either ssDNA binding or formation of a heparin-resistant E⁷⁰-forked complex, with the k_obs of open complex formation. No significant correlation was observed between the fraction of ssDNA bound (Fig. 5) and k_obs (Fig. 3) (R² = 0.03). However, in searching for a correlation between k_obs for the various mutant E⁷⁰ and their ability to form a heparin-resistant complex with the short fork, a peculiar but striking difference in the behavior of E⁷⁰ bearing alanine substitutions for basic and aromatic amino acids in region 2 came to light, as shown in Fig. 8. If the entire data set is considered, a reasonable (R² = 0.7) correlation is observed between the k_obs and the % forked DNA bound in a heparin-resistant complex. However, the correlation was much better (R² = 0.94) when only substitutions of alanine for basic residues were considered; the corresponding linear least squares fit is displayed in Fig. 8. The aromatic residues are clearly off the line. The points for Y433A, F427A, and Y425A (e, f, and g, respectively) lie above the plot (i.e. compared with the substitutions for basic amino acids, they bind better to the forked DNA than expected based on their value of k_obs for open complex formation). The points for the multiply substituted sigma factors (a, b, and c) as well as for Y430A (d) show the opposite behavior. There was no a priori reason for expecting a correlation between the kinetic data for open complex formation at promoters and the equilibrium data for the extent of short fork binding. However, the fact that one is observed indicates that the differences in dissociation rates (or half-lives) we observe for the forked DNAs (see Fig. 7) might be small compared with the differences that exist in the association rate constants. Then the fork binding data would essentially reflect relative rates of formation of complexes.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Correlation between kobs for open complex formation (Fig. 3) and the extent of short fork binding (Fig. 7). The line shown is the linear least squares fit to the data for the E70-bearing alanine substitutions for basic amino acids (filled squares; 1-6 are for substitutions at positions 418, 414, 436, 423, 426, and 422, respectively). The open triangles represent data points for E70-bearing alanine substitutions for aromatic amino acids in region 2 of 70 (a-c are for YYW, FYW, and YW, and d-g are for substitutions at positions 430, 433, 427, and 425, respectively). Open circle, wt E70.

Some time ago, Record and colleagues (2) as well as Buc and McClure (1) showed that formation of an open complex proceeds through at least two intermediates.

Here R represents the RNA polymerase holoenzyme and P the promoter. R·P_c1 is the first sequence-specific complex formed, where the strands have not yet separated; it is often referred to as the closed complex. R·P_c2 is the second intermediate, where E⁷⁰ may have undergone a conformational change and the nucleation of strand separation may have taken place, perhaps by the flipping of the 11A out of the helix (14). We interpret our results in terms of the basic and aromatic residues being involved in the different steps of Scheme 1. We have shown previously that the aromatic amino acids of ⁷⁰ region 2.3 were probably involved in the second step of Scheme 1, the interconversion between R·P_c1 and R·P_c2 (11). In agreement with this interpretation, the results obtained here show that E⁷⁰, bearing multiple substitutions for aromatic amino acids, is deficient in binding ss and short fork DNA as well as in stabilizing E⁷⁰-short fork complexes. Based on the results presented here, we conclude that the basic amino acid residues would facilitate the formation of R·P_c1. This conclusion is supported by the comparison of affinities with which E⁷⁰ bearing alanine substitutions for these residues bound short fork DNA.² It also is consistent with the failure to observe a heparin-sensitive short footprint for these mutant E⁷⁰ (data not shown) as was observed for the YYW and FYW E⁷⁰ (11).

Residues Lys⁴¹⁴, Lys⁴¹⁸, Tyr⁴³⁰, and Trp⁴³³ appear particularly important for progressing through the individual steps shown in Scheme 1. Our results indicate that Lys⁴¹⁴, Lys⁴¹⁸, and Tyr⁴³⁰ are vital in the formation of heparin-resistant E⁷⁰-forked DNA complexes; in addition, Tyr⁴³⁰ plays a crucial role in the stabilization of these complexes. The effects of alanine substitutions for Lys⁴¹⁴, Lys⁴¹⁸, and Trp⁴³³ on open complex formation are evident at both 20 and 37 °C, whereas the effect of substitution for Tyr⁴³⁰ apparently can be masked partially at 37 °C but not at 20 °C. These four amino acid residues are close to each other in ⁷⁰; they are located within a sphere with a radius of about 5 Å in the structure of ⁷⁰ (16). We propose that, together, they participate in the nucleation of strand separation. The roles of the two basic amino acids would be to hold the promoter DNA in the proper orientation and allow the aromatic amino acids to nucleate the strand separation process, likely by flipping the 11A out of the helix by a mechanism that is not yet fully understood. It has been proposed that the aromatic rings of residues 430 and 433 would sandwich the 11A in between them (14). This model is unlikely to be entirely correct, as our findings clearly indicate that alanine substitutions for the two residues do not behave similarly in all assays. The substitution for Tyr⁴³⁰ has a much more pronounced effect on forked DNA binding to E⁷⁰, whereas substitution for Trp⁴³³ has a greater effect on k_obs for open complex formation at 37 °C. Also, it is evident from the results presented in Fig. 8 that Y430A behaves differently from the other single substitutions for aromatic amino acids we examined. One possibility for reconciling the findings is that Trp⁴³³ participates in "forcing" the flipped base out of a DNA duplex, whereas Tyr⁴³⁰ would interact with the flipped out base as provided either by the forked DNA or subsequent to the action of Trp⁴³³ on duplex DNA.

It is also unlikely that three electron-rich rings, such as those of Tyr⁴³⁰, 11A, and Trp⁴³³, would engage in the formation of a sandwich. Such a sandwich has been proposed for the human 3-methyladenine DNA glycosylase (25), but the DNA base in that case is alkylated and electron-deficient. It is more likely that the putatively flipped out 11A would partially overlap one of the aromatic residues, perhaps the tyrosine at 430, so that electron-deficient regions of one would be over the electron cloud of the other. This would also be more consistent with the structural and mutagenesis data that have been reported for flipped out bases, which are found in close proximity to just one aromatic amino acid residue. This is a tryptophan in the case of the E. coli repair enzyme AlkA (26) and the Tn5 transposon (27), a tyrosine for human 3-methyladenine DNA glycosylase and B. subtilis DNA polymerase I (25, 28), and a phenylalanine for N6-adenine DNA methyltransferase (29) and E. coli and human uracil DNA glycosylase (30, 31). Another aromatic residue could be involved in the above mentioned forcing out of the flipped base or could be interacting edge-on with it, like tyrosines 162 and 159, respectively, of human 3-methyladenine DNA glycosylase (25) and here possibly Trp⁴³³ and/or Trp³³⁴.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 31808 (to P. L. dH.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The first two authors contributed equally to this work.

^§ To whom correspondence should be addressed: Dept. of Biochemistry, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4935; Tel.: 216-368-3684; Fax: 216-368-4544; E-mail: pld2@po.cwru.edu.

Published, JBC Papers in Press, July 6, 2001, DOI 10.1074/jbc.M105027200

² L. Tsujikawa, O. Tsodikov, and P. L. deHaseth, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: E, RNA polymerase core enzyme; E, RNA polymerase holoenzyme; E⁷⁰, RNA polymerase holoenzyme reconstituted from core and purified ⁷⁰; EMSA, electrophoretic mobility shift assay; oligo, oligodeoxyribonucleotide; ss, single-stranded; wt, wild type.

	REFERENCES

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