Architecture of a Complex between the sigma 70 Subunit of Escherichia coli RNA Polymerase and the Nontemplate Strand Oligonucleotide
LUMINESCENCE RESONANCE ENERGY TRANSFER STUDY*

Ewa Heyduk and Tomasz HeydukDagger

From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104

    ABSTRACT
Top
Abstract
Introduction
References

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma 70 holoenzyme. Five single reactive cysteine mutants of sigma 70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma 70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma 70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma 70 is located in an alpha -helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.

    INTRODUCTION
Top
Abstract
Introduction
References

Transcription initiation in Escherichia. coli involves two essential steps: (i) initial promoter recognition by RNA polymerase (RNAP1) and (ii) melting of DNA duplex in the vicinity of transcription start site (1-5). The simplest two-step model describing transcription initiation (Eq. 1) involves a rapid formation of a labile "closed" complex that in a second step isomerizes to a stable "open" complex.
<UP>RNAP</UP>+<UP>DNA → RNAP · DNA<SUB>closed</SUB> → RNAP · DNA<SUB>open</SUB></UP> (Eq. 1)
In the open complex 10-15 base pairs of DNA duplex become single-stranded. This DNA duplex melting in the case of E. coli RNAP occurs spontaneously, therefore the energetic cost of duplex melting must be offset by some favorable RNAP-promoter interactions.

The E. coli RNA polymerase holoenzyme is a multisubunit enzyme (subunit composition alpha 2beta beta 'sigma ) (1). sigma 70 (the primary sigma  subunit) is the RNAP subunit thought to be responsible for the initial recognition of promoter DNA (1-6). Sequence homology between the conserved region 2.3 of sigma 70 and eukaryotic single-stranded DNA (ssDNA)-binding proteins was used as a basis of the proposal that sigma 70 subunit could also be actively involved in the promoter melting reaction through binding of ssDNA of the open complex (7). This favorable ssDNA-sigma 70 interaction could reduce the energetic cost of DNA melting and facilitate open complex formation. The data from several laboratories provided experimental evidence in support of this proposal. The sigma 70 subunit was shown to bind ssDNA (8-16) or single-stranded bubbles within the DNA duplex (17, 18). The binding was specific for the nontemplate DNA strand (9-16). The ssDNA binding site of sigma 70 is most likely located in the conserved region 2.3 of the sigma 70 subunit (13, 14, 16, 19-21). The ssDNA binding activity of sigma 70 is regulated allosterically through binding to the core RNAP (16). Free sigma 70 binds ssDNA weakly and does not discriminate between the template and nontemplate sequences. Binding of sigma 70 to the core reduces the affinity of sigma 70 to template ssDNA and increases the affinity to nontemplate ssDNA resulting in a ~200-fold difference in the affinity between nontemplate and template ssDNA (16). All of the above properties of sigma 70 are consistent with its active role in the promoter melting reaction.

Region 2.3 of sigma 70 is thought to be a location of the ssDNA binding site of sigma 70. However, the architecture of the ssDNA-sigma 70 complex and the structural determinants of the high selectivity for nontemplate ssDNA binding are not known. Therefore, in this work we used luminescence energy transfer (LRET) (22-25, 31, 32) distance measurements to determine localization of nontemplate strand oligonucleotide bound to sigma 70 with respect to several functional domains of the protein.

    EXPERIMENTAL PROCEDURES

Materials-- Cy5, monosuccinimidyl ester, was purchased from Amersham Pharmacia Biotech. Succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS) was from Boehringer Mannheim. Oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). All other chemicals were of the highest purity commercially available. Core RNAP was purified from E. coli K12 cells (obtained from the University of Alabama fermentation facility) using the method of Burgess and Jendrisak (29).

Preparation of DTPA-AMCA(5)-Maleimide-- 5 mg of AMCA-NHS was dissolved in 250 µl of DMF, and 50 µl of 1 M ethylenediamine HCl (pH 7.0) was added. The mixture was incubated for 1 h at room temperature, and another 50 µl of 1 M ethylenediamine HCl was added followed by incubation for 1 h at room temperature. The mixture was diluted to ~4 ml with buffer A (25 mM triethylammonium acetate buffer (pH 7.0) containing 2% acetonitrile) + 300 µl of buffer B (buffer B is buffer A with 95% acetonitrile). A small amount of a white precipitate was removed by a centrifugation, and the sample was loaded on a fast protein liquid chromatography reverse phase column (HR10/10 column (Amersham Pharmacia Biotech)) packed with Resource 15RPC (Amersham Pharmacia Biotech).The column was eluted at 3 ml/min with 100 ml of 0-50% buffer B gradient. Fractions containing the adduct of 7-amino-4-methyl coumarin-3-acetic acid and ethylenediamine eluting at about 14% B were pooled (7.5 ml) and lyophilized. Dried fractions were dissolved in 500 µl of DMF, and 5 mg of succinimidyl ester of maleimidylpropionic acid dissolved in 100 µl of DMF was added. The mixture was incubated for 1 h at room temperature, diluted to ~ 5 ml with 5% buffer B, and loaded onto a Resource 15RPC column. The column was eluted at 3 ml/min with 100 ml of 0-50% buffer B gradient. Fractions containing the AMCA-maleimide eluting at about 24% buffer B were pooled (7.5 ml) and lyophilized. Dried fractions were dissolved in 500 µl of DMF, and 20 mg of DTPA anhydride was added. The mixture was incubated for 1 h at room temperature, diluted to 5 ml with buffer A, and run on a Resource 15RPC column as described above. Fractions containing DTPA-AMCA-maleimide eluting at ~15% buffer B were pooled, dispensed to Eppendorf tubes such that each tube contained 0.1 µmol of the chelate, and dried. The yield of a purified product was 10-20%.

Single-cysteine Mutants of sigma 70-- The preparation of single-cysteine mutants of sigma 70 is described elsewhere (27). Briefly, three endogenous cysteine residues of sigma 70 (cysteines 132, 291, and 295) were replaced with Ser residues using site-directed mutagenesis. Single Cys residues were then introduced into the desired locations using single amino acid replacements, with the exception of [1Cys]sigma 70, in which the cysteine residue was inserted between the initiating Met and the second residue of the protein. The following single-cysteine mutants of sigma 70 were used in LRET experiments: [1C]sigma 70, [A59C]sigma 70, [S366C]sigma 70, [S442C]sigma 70, and [R596C]sigma 70. Single-cysteine mutants of sigma 70 were expressed and purified as described before (27). Transcriptional activity of single-cysteine mutants of sigma 70 compared with the wild type sigma 70 was: 109% ([1C]sigma 70), 83% ([A59C]sigma 70), 64% ([S366C]sigma 70), 95% ([S442C]sigma 70), and 100% ([R596C]sigma 70) (27).

Fluorochrome-labeled Oligonucleotides-- In all experiments a 12-nt oligonucleotide (TCGTATAATGTG) corresponding to positions -15 to -4 of the lacUV5 promoter nontemplate strand was used. The Cy5 fluorochrome was attached to the 5' end by first adding a 5'-aliphatic amino group through a postsynthetic modification of the oligonucleotide with ethylenediamine (28), which results in a two-carbon linker between the 5'-phosphate and the reactive amine. The 5'-amino-containing oligonucleotide was then reacted with ~ 1 mM succinimidyl ester of Cy5 for 2-4 h at room temperature in 0.1 M sodium bicarbonate buffer (pH 8.3). The excess of Cy5 was removed on a G-25 spin column (Amersham Pharmacia Biotech), and the labeled oligonucleotide was purified from unlabeled DNA using a reverse phase high performance liquid chromatography column as described previously (26). To attach Cy5 fluorochrome to the 3'-end of the oligonucleotide a 3'-amino group was introduced during oligonucleotide synthesis using a three-atom linker. The reaction of the 3'-amino-containing oligonucleotide with Cy5 and the purification of labeled DNA were performed as described for 5'-amino-modified oligonucleotide. The concentration of the oligonucleotides was determined spectrophotometrically using absorbance at 260 nm corrected for the contribution due to Cy5 dye (26).

Donor Fluorochrome-labeled sigma 70-- Samples of each single-cysteine mutant of sigma 70 (0.5-1.0 mg) were precipitated with 60% ammonium sulfate by the addition of the appropriate volume of saturated ammonium sulfate solution. The protein pellet was collected by centrifugation and dissolved in 75 µl of 50 mM Tris (pH 8.0), 1 mM EDTA, 5% glycerol, and 6 M GdHCl buffer. Dithiothreitol was added to a final concentration of 0.5 mM, and mixtures were incubated for 1 h at room temperature. Dithiothreitol was removed by a Microspin G-50 column (Amersham Pharmacia Biotech) equilibrated with the above buffer. DTPA-AMCA-maleimide was added to a final concentration of 0.5-1.0 mM, and the reaction was allowed to proceed for 1 h at room temperature. The excess of unreacted DTPA-AMCA-maleimide was removed by Microspin G-50 column equilibrated with 50 mM Tris (pH 8.0), 1 mM EDTA, 5% glycerol, and 6 M GdHCl buffer. The eluate from the G-50 column was diluted to ~0.75 ml with the above buffer, dialyzed first against the same buffer for few hours and next to 50 mM Tris (pH 8.0), 5% glycerol buffer overnight with three changes of 100 ml of the buffer. Refolded modified sigma 70, after a 30-min incubation with 10 µM EDTA and 10 µM EuCl3, was mixed with purified core RNAP in a 1:1 molar ratio and incubated for 30 min at 4 °C. The reconstituted holoenzyme was purified from unbound labeled sigma 70 on a fast protein liquid chromatography Superdex-200 size exclusion column (Amersham Pharmacia Biotech). For experiments with free donor-labeled sigma 70, the protein after refolding through dialysis was purified further on Superdex-200 column.

LRET Measurements-- All LRET experiments were performed in a 120-µl cuvette in 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 5% glycerol buffer at 25 °C. The concentration of the holoenzyme used was 25-50 nM. Luminescence decays of donor fluorochrome-labeled holoenzyme were recorded in the presence and absence of acceptor-labeled oligonucleotides (0-250 nM). Luminescence lifetime measurements were performed on a laboratory-built two-channel spectrofluorometer with a pulsed nitrogen laser (LN300, Laser Photonics, Orlando, FL) as an excitation source (26). Donor emission was observed at 617 nm and acceptor emission at 670 nm. Decays of donors in the absence of acceptor were fitted to a single-exponential equation,
I(t)=&agr;<UP>exp</UP>(<UP>−</UP>t/&tgr;) (Eq. 2)
where alpha  is the amplitude and tau  is the luminescence lifetime, respectively. Decays of donors in the presence of acceptor and decays of sensitized acceptor emission were fitted to a three-exponential equation (see "Results"). Donor and sensitized acceptor decay curves were fitted simultaneously using global nonlinear regression with Scientist (Micromath Scientific Software, Salt Lake City, UT) to the following set of equations:
I<SUB>d</SUB>(t)=<LIM><OP>∑</OP></LIM> &agr;<SUB>i,d</SUB> <UP>exp</UP>(<UP>−</UP>t/&tgr;<SUB>i</SUB>) (Eq. 3)
I<SUB>a</SUB>(t)=<LIM><OP>∑</OP></LIM> &agr;<SUB>i,a</SUB> <UP>exp</UP>(<UP>−</UP>t/&tgr;<SUB>i</SUB>) (Eq. 4)
where Id(t) and Ia(t) are luminescence intensity of donor and sensitized acceptor, respectively; alpha i,d and alpha i,a are amplitudes of the ith component in donor and sensitized acceptor decay, respectively; and tau i is the lifetime of the ith component. Such global fitting is possible because of unique properties of the europium chelate Cy5 donor-acceptor pair (long microsecond lifetime of the donor and nanosecond lifetime of the acceptor). The decay of sensitized acceptor in microsecond time scale under these conditions occurs with lifetimes of the donor engaged in energy transfer with the acceptor (26, 31-33). Thus, decays of the donor and sensitized acceptor are described by the same lifetimes and different amplitudes (Equations 3 and 4).2 The ability to analyze donor- and sensitized-acceptor decay data by global fitting to Equations 3 and 4 is an important advantage of using europium chelate in LRET experiments. It improves very significantly the precision and the confidence of lifetime determination in the presence of energy transfer.

The energy transfer (E) between europium chelate-labeled sigma 70 and Cy5-labeled oligonucleotide was calculated from measurements of luminescence lifetime of a donor in the absence (tau d) and in the presence of acceptor (tau da).
E=1−&tgr;<SUB>da</SUB>/&tgr;<SUB>d</SUB> (Eq. 5)
The distances between the donor and the acceptor were calculated according to Förster theory (22)
R<SUP>6</SUP>=R<SUP>6</SUP><SUB>0</SUB>(1−E)/E (Eq. 6)
where R is a distance between a donor and an acceptor, and R0 is a distance at which the energy transfer is 0.5. The R0 (55 Å) was calculated as described previously using assumptions described by Selvin and Hearst (31). One of these assumptions is that the orientation factor (kappa 2) value of 2/3 (completely random orientation of donors and acceptors (22-25)) could be used in calculating R0. This assumption is justified by two factors: long lifetime of the donor and multiple transition dipole moments of the lanthanide (26, 31-33). The long donor lifetime increases the probability that a donor and an acceptor will rotate to all possible orientations during the donor excited state lifetime. Multiple transition dipole moments result in depolarization of donor emission even if the donor is completely immobile.

    RESULTS

Acceptor-labeled 12-nt Oligonucleotide and Donor-labeled sigma 70-- Fig. 1, A and B, shows the structure of the two acceptor fluorochrome-labeled oligonucleotides used in this work. The 12-nt oligonucleotide sequence used corresponds to position -15 to -4 of the lacUV5 nontemplate strand sequence. We have shown previously that sigma 70 in a RNAP holoenzyme is capable of binding this oligonucleotide ~200-fold better than the template or random sequence 12-nt oligonucleotide (16). The acceptor fluorochrome (Cy5) was attached to the 5'-end (Cy5-5'-NT) or to the 3'-end (NT-3'-Cy5) of the oligonucleotide, respectively. Fig. 1C shows an absorbance spectrum of the purified Cy5-5'-NT. The characteristic absorbance peaks caused by DNA (at 260 nm) and Cy5 (at 647 nm) are apparent. Using this spectrum we estimated that labeled oligonucleotide contained ~1 mol of Cy5 dye/mol of the oligonucleotide. Essentially identical spectrum and degree of modification were observed for NT-3'-Cy5 (not shown).


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Fig. 1.   Structure of acceptor-labeled 12-nt nontemplate oligonucleotides. The acceptor (Cy5) was attached to the 5'-end (panel A) or to the 3'-end (panel B) of the oligonucleotide. Panel C, absorption spectrum of a purified labeled Cy5-5'-NT oligonucleotide. The spectrum for NT-3'-Cy5 oligonucleotide was essentially the same.

Fig. 2A shows the structure of the fluorescence donor molecule used in our studies. The europium ion coordinated by the DTPA moiety of the probe is a luminescent component of the donor probe (26). We used a europium chelate for LRET measurements because, as shown recently, these probes offer several important advantages when used as donors in LRET measurements compared with classical organic dye fluorescence probes (26, 31-33). The fluorescence donors were incorporated into the specific sites of sigma 70 protein through chemical modification of unique cysteine residues placed in different structural domains of the protein (Fig. 2B). Cys-1 and Cys-59 are in conserved region 1, which was shown to be involved in the autoinhibition of promoter DNA binding in the free sigma 70 (34, 35). Cys-366 is located in a nonconserved region of the protein near sequences thought to be important for core RNAP binding (36, 37). Cys-442 is in region 2.4, responsible for -10 promoter DNA sequence recognition, and is adjacent to region 2.3, thought to be involved in nontemplate ssDNA binding (37-39). Cys-596 is in region 4.2, which was shown to be involved in -35 promoter DNA sequence recognition (40). Fig. 2C shows an example of the absorption spectrum of the purified donor-labeled sigma 70 ([S366C]sigma 70). Characteristic peaks caused by protein (at ~280 nm) and DTPA-AMCA (at ~328 nm (26)) were observed. Using this spectrum we estimated that the labeled protein contained ~1 mol of the AMCA-DTPA/mol of the protein. The degree of labeling for other donor-labeled sigma 70 proteins was 0.5-1.0 mol of AMCA-DTPA/mol of protein.


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Fig. 2.   Panel A, structure of europium chelate used as a donor in FRET measurements. Panel B, localization of single-reactive cysteine residues in the primary structure of sigma 70 protein. Conserved regions of the protein (43) are indicated by boxes. Panel C, absorption spectrum of the purified donor-labeled [S366C] sigma 70 protein. Spectra for other single-cysteine mutants of sigma 70 were very similar.

LRET Measurements-- Figs. 3 and 4 show representative examples of LRET data for the donor-labeled sigma 70 reconstituted with the core RNAP, in the presence and absence of acceptor-labeled oligonucleotides. The data shown in Figs. 3 and 4 are for the donor-labeled [S366C]sigma 70 and 12-nt oligonucleotide labeled with Cy5 at the 5'-end (Cy5-5'-NT). The results for other donor-labeled sigma 70 and Cy5-5'-NT or NT-3'-Cy5 oligonucleotides were qualitatively similar. Only the extent of LRET observed was different with different combinations of labeled sigma 70 and labeled oligonucleotides. LRET data for all of these combinations are summarized in Tables II and III.


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Fig. 3.   Decay curves of donor-labeled [S366C]sigma 70. Panels A and B, donor-labeled [S366C]sigma 70 reconstituted with the core RNAP; panels C and D, free donor-labeled [S366C]sigma 70. Circles represent the data obtained in the absence of DNA, and triangles represent the data obtained in the presence of 100 nM acceptor-labeled Cy5-5'-NT DNA. Panels A and C represent donor decay curves collected using emission at 617 nm, and panels B and D represent sensitized acceptor decays collected at 670 nm. Solid lines represent best fits of experimental data to decay equations as described under "Experimental Procedures."


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Fig. 4.   Decay curves of donor-labeled [S366C]sigma 70 reconstituted with the core RNAP in the presence and absence of the excess of unlabeled oligonucleotides. Panel A: circles, [S366C]sigma 70 holoenzyme; triangles, [S366C]sigma 70 holoenzyme + 50 nM Cy5-5'-NT oligonucleotide. Panel B: circles, [S366C]sigma 70 holoenzyme; triangles, [S366C]sigma 70 holoenzyme + 50 nM Cy5-5'-NT and 1 mM unlabeled 12-nucleotide nontemplate oligonucleotide. Panel C: circles, [S366C]sigma 70 holoenzyme; triangles, [S366C]sigma 70 holoenzyme + 50 nM Cy5-5'-NT and 1 mM unlabeled 12-nucleotide nontemplate randomized sequence oligonucleotide.

In the absence of acceptor-labeled oligonucleotide, luminescence decays of donor-labeled sigma 70 reconstituted with the core RNAP were monoexponential (Fig. 3A). In the presence of acceptor-labeled oligonucleotide, decays of donor-labeled sigma 70 were no longer single exponential, and the presence of faster decaying component(s) was obvious (Fig. 3A). The decay of the donor-labeled holoenzyme in the presence of acceptor-labeled oligonucleotide could be fitted to a three-exponential decay function. At any given concentration of acceptor-labeled oligonucleotide, we expected in solution an equilibrium mixture of RNAP-oligonucleotide complex (capable of LRET) and free RNAP (incapable of LRET). In accordance with this expectation, the slowest decay time (tau 3) observed in the presence of acceptor-labeled oligonucleotide was very similar to the decay time observed in the absence of acceptor, and its amplitude decreased with the increase of oligonucleotide concentration (Table I). The faster decaying component(s) were thus interpreted to be caused by LRET between donor-labeled sigma 70 and acceptor-labeled oligonucleotide. Three observations support this interpretation. First, in the presence of unlabeled 12-nt nontemplate oligonucleotide, decay of the donor remained monoexponential with the lifetime very similar to the one in the absence of any DNA (not shown). Second, the appearance of a fast decaying component(s) in the presence of acceptor-labeled oligonucleotide was correlated with the appearance of a large sensitized emission signal of the acceptor (Fig. 3B). Third, the amplitude of the fast component(s) increased with the increase of acceptor-labeled oligonucleotide concentration, whereas the lifetimes of the fast and slow components remained constant (Table I).

                              
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Table I
Amplitudes of fast and slow decay components as a function of 12-nucleotide oligonucleotide concentration
The data in the table are for Cy5-5'-NT 12-nucleotide oligonucleotide and the 50 nM holoenzyme reconstituted with sigma 70 labeled with fluorescence donor at Cys-366.

Two lifetimes (tau 1 and tau 2) were necessary to describe the fast decaying portion of the decay curve adequately. There are several possible interpretations for the two fast decaying components observed in the presence of acceptor. They could be a result of two populations of species capable of LRET; for example, the oligonucleotide could bind to the primary binding site and, to a lesser degree, to a secondary site. The appearance of two fast decaying components in LRET with lanthanide chelates was observed previously, and the very fast component was interpreted to be an instrumental artifact (30-32). In cases where the donor-acceptor distance was large, the two fast lifetimes could be resolved. For example, in the case of donor-labeled [R596C]sigma 70 and NT-3'-Cy5 DNA these lifetimes were: tau 1 = 66 µs (9% of total fast component amplitude) and tau 2 = 412 µs (91% of total fast component amplitude). However, when the distance between the donor and acceptor was smaller, the values of these two lifetimes became correlated and could not be well resolved. Thus, for all LRET calculations presented here we used the weighted average of two fast decaying components, avoiding the arbitrary decision of which lifetime component to use. The impact of the mode of LRET calculations on the results was small because the distances obtained using the average of tau 1 and tau 2 were very similar to distances calculated using only tau 2 (the average difference in distances between these two modes of calculation was only 4 ± 2 Å).

LRET between the donor-labeled sigma 70 and acceptor-labeled 12-nt nontemplate strand oligonucleotide was specific only for sigma 70 reconstituted with the core RNAP. Free sigma 70 showed no evidence of significant energy transfer in the presence of 100 nM oligonucleotide (Fig. 3, C and D). Donor decays in the presence or absence of acceptor-labeled DNA were monoexponential (Fig. 3C), and essentially no sensitized emission of the acceptor was observed (Fig. 3D). At the same concentration of labeled oligonucleotide a very efficient LRET was observed in the case of holoenzyme (Fig. 3, A and B). These observations are consistent with and further confirm our previous report that the specificity for binding the nontemplate single-stranded oligonucleotide is induced in sigma 70 by an interaction with the core RNAP (16).

Competition experiments were used to determine the specificity of LRET in the nontemplate strand oligonucleotide-holoenzyme complex (Fig. 4). In the presence of 50 nM acceptor-labeled nontemplate strand oligonucleotide an efficient LRET was observed as indicated by a much faster decay of the donor compared with the decay in the absence of DNA (Fig. 4A). This efficient LRET could be eliminated by the addition of excess unlabeled 12-nt nontemplate strand oligonucleotide as indicated by almost overlapping decay curves observed in the absence of any DNA and in the presence of 50 nM acceptor-labeled and 1 µM unlabeled nontemplate oligonucleotide (Fig. 4B). In contrast, excess unlabeled nontemplate randomized sequence oligonucleotide had essentially no effect, and the efficient LRET was still observed, as indicated by a much faster decay observed in the presence of 50 nM acceptor-labeled nontemplate oligonucleotide and 1 µM unlabeled nontemplate randomized sequence oligonucleotide (Fig. 4C). The nontemplate randomized sequence oligonucleotide (TTGATATCGTAG) had the same base composition but a sequence different from that of the nontemplate strand oligonucleotide. Based on the results presented in Figs. 3 and 4 and Table I we concluded that LRET signals observed for donor-labeled sigma 70 and acceptor-labeled nontemplate strand oligonucleotides are the property of a specific complex between the holoenzyme and nontemplate ssDNA. The distances calculated from these LRET measurements provide information regarding the architecture of this complex.

Architecture of sigma 70-Nontemplate ssDNA Complex-- Results of LRET measurements with all five single-cysteine mutants of sigma 70 and nontemplate 12-nt oligonucleotides with acceptors at the 3'- or 5'-end are summarized in Tables II and III. A wide range of energy transfer efficiencies (from 0.37 to >0.99) was observed. The range of distances corresponding to these energy transfer efficiencies was from 60 Å to < 25 Å. For several residues of sigma 70 very significant differences between the distance to 5' and 3' of the oligonucleotide were found. Region 2.4 (Cys-442) was found to be the closest to the oligonucleotide bound to sigma 70. It appears also that the 5'-end of the bound oligonucleotide was much closer (<25 Å) to residue 442 than the 3'-end (35 Å). Residue 596 was the farthest from the bound oligonucleotide, and this residue seems to be located almost at the same distance from the 5'- and 3'-ends of the oligonucleotide. Also, the NH2-terminal cysteine was found at an approximately equal distance from the 5'- and 3'-ends of the oligonucleotide. Residue 59 was found closer to the 5'-end of the oligonucleotide, and residue 366 was found closer to the 3'-end.

                              
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Table II
LRET distance measurements between donor-labeled single cysteine mutants of sigma 70 and 5'-end of 12-nucleotide nontemplate DNA (NT-5'-Cy5)

                              
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Table III
LRET distance measurements between donor-labeled single cysteine mutants of sigma 70 and 3'-end of 12-nucleotide nontemplate DNA (Cy5-3'-NT)

Using distances determined by LRET, three-dimensional models of relative localization of different domains of sigma 70 could be built. An example of such a model superimposed on the crystal structure of sigma 70 fragment is shown in Fig. 5. Building these models allowed an indirect determination of several additional distances between the sites in the complex (Table IV). Because there were not enough distance constraints to determine all possible distances uniquely, an analysis of a relative precision of these indirect distance determinations was performed. A set of 25 independent models fulfilling distance constraints from LRET experiments was built, starting each from randomly "scrambled" initial distances between the sites in the complex. In each of these models all possible distances were then measured, and the mean and standard deviation were calculated (Table IV). The standard deviation can thus be used as a convenient measure of a relative precision of these indirect distance estimations. Inspection of the data presented in Table IV shows that two sets of distances can be identified easily: those very poorly defined (standard deviation > 20 Å) and those whose precision is good enough (standard deviation < 10 Å) for use in discussing the architecture of the complex. Distances with relatively high precision of estimation were 5' of the oligonucleotide to 3' of the oligonucleotide, residues 1-442, 9-442, 366-442, and 442-596. We have recently measured several interdomain distances in the holoenzyme,3 and these distances appear to be in general agreement with the distances calculated by model building (Table IV). Additionally, using these directly determined interdomain distances we attempted to obtain a better estimate of 5' iff  442 distance by building models that included this distance as a variable. This distance could not be determined by LRET measurements because the donor and acceptor were too close (Table II). A distance of ~14 Å was obtained from model building, consistent with LRET results (Table II).


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Fig. 5.   Schematic model representing the relative localization of different domains of sigma 70 with respect to 5'- and 3'-ends of the RNAP-bound nontemplate 12-nt oligonucleotide. Panels A and B correspond to two different views of the model differing in ~ 90° rotation around the x axis. The model was built as described under "Experimental Procedures" and was aligned with the crystal structure of the sigma 70 fragment (37) such that positions 366 and 442 (present both in the model and in the structure) were superimposed. The model shown is one of many possible configurations fulfilling distance constraints (Tables II-IV) and is shown only for visual illustration of the data presented in these tables. The size of the spheres is proportional to a distance to the viewer. The model was built using distance constraints routine of ChemSite (Pyramid Learning, Stanford, CA), and the figure was produced using Ribbons (45).

                              
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Table IV
Calculated distances between sites in holoenzyme-oligonucleotide complex using distance constraints from LRET measurements


    DISCUSSION

We have determined the distances between several sites in the sigma 70 and the 5'- or the 3'-end of 12-nt nontemplate strand oligonucleotide in complex with RNAP holoenzyme. The measured distances allowed us to build a model describing a three-dimensional architecture of the oligonucleotide-RNAP complex. Several conclusions regarding the architecture of the complex can be made.

The oligonucleotide binds to RNAP apparently in a preferred orientation. In principle, the oligonucleotide could bind to its binding site either in 5' right-arrow 3' or 3' right-arrow 5' orientation. If the binding could occur equally well in either orientation, the apparent distances measured between sites in sigma 70 and the 5'- or the 3'-end of the oligonucleotide should be the same. We observed, however, that for several sites in sigma 70 the distances between the 5'-end and the 3'-end were very significantly different (Tables II and III), showing that the oligonucleotide was bound in one preferred orientation. Such preference for a specific orientation may be an additional mechanism by which RNAP in the open complex could discriminate between template and nontemplate strands in the transcription bubble. Previous binding experiments with oligonucleotides corresponding to the -10 region of the nontemplate and the template strand showed that RNAP holoenzyme could bind nontemplate sequence oligonucleotides ~200-fold better then the template sequence oligonucleotides (16). Oligonucleotides can freely assume any orientation when they bind to the ssDNA binding site of sigma 70. The situation will be different in the open complex when ssDNA strands in the -10 region have restricted mobility. Thus, if the nontemplate strand in the open complex is in a correct orientation for binding to the ssDNA binding site of sigma 70, the template strand will be forced to be in the opposite, unfavorable for the binding orientation. Thus, the two different mechanisms for discrimination between single-stranded nontemplate and single-stranded template strands in the -10 region of promoter DNA are apparently being used by sigma 70. One mechanism is the sequence specificity of the binding, the other mechanism is the strand polarity specificity. This dual mode of discrimination employed by sigma 70 seems to be well suited for the tasks that the ssDNA binding site of sigma 70 needs to perform: selective binding of the -10 sequences and selective binding of the nontemplate strand.

Region 2.4 (Cys-442) was found to be the closest to the oligonucleotide bound to sigma 70. This is consistent and confirms the proposals that the ssDNA binding site of sigma 70 is localized in region 2.3 (13, 14, 16, 19-21) because this region is adjacent to region 2.4 and is located in the same alpha -helix (helix 14 (37)). It appears also that the 5'-end of the bound oligonucleotide was much closer (<25 Å) to residue 442 than the 3'-end was (39 Å). Thus, the relative orientation of the bound oligonucleotide with respect to helix 14 appears to be 5' right-arrow COOH terminus of the helix. Such an orientation is consistent with the proposed model of nontemplate ssDNA-sigma 70 interaction based on the crystal structure of the sigma 70 fragment (37). It is also consistent with data relating mutations in sigma 70 and mutations in the -10 region of promoter DNA. Based on these studies it was proposed that residues 437 and 440 are involved in recognition of position -12, whereas residue 441 is involved in recognition of position -13 (38, 39, 41, 42). Such an alignment of bases in the -10 region and amino acids in helix 14 is consistent with a 5' right-arrow COOH terminus alignment of helix 14 and the nontemplate oligonucleotide in a holoenzyme-oligonucleotide complex. However, an opposite orientation of the nontemplate strand with respect to helix 14 was also proposed (44). The reasons for this discrepancy are not clear.

Cys-596 is in region 4.2 of sigma 70, which was suggested to bind the -35 region of promoter DNA (40). Assuming a simple linear arrangement of -10 and -35 DNA sequences and protein domains involved in binding of these sequences, it could be expected that the 5'-end of the bound oligonucleotide should be much closer to residue 596 than the 3'-end is. Data in Tables II and III show that this was not observed. In contrast, residue 596 seems to be located almost at the same distance from the 5'- and 3'-ends of the oligonucleotide. This suggests that the orientation of the bound oligonucleotide with respect to residue 596 is as illustrated in Fig. 5, i.e. it is more or less perpendicular, not parallel, to the line joining regions 2.4 and 4.2. This observation suggests that promoter DNA in the open complex is not straight, and thus formation of the open complex involves significant deformation of DNA.

Based on LRET distance measurements it was possible to estimate indirectly with reasonable precision several other distances in the oligonucleotide-RNAP complex. The predicted distance between the 5'- and 3'-ends of the oligonucleotide was 32 ± 7 Å, a distance somewhat shorter than expected for a linear 12-nt DNA, consistent with the deformation of DNA in the open complex. The predicted distance between residues 366 and 442 was found to be 38 ± 6 Å. This distance is comparable, within the error of estimation, to the distance between these two residues (35 Å) observed in the crystal structure of sigma 70 fragment (37). The agreement of this predicted distance with the crystal structure of the sigma 70 fragment provides an additional validation of distances measured by LRET. The predicted distance between residues 442 and 596 was 58 ± 6 Å. Residues 442 and 596 are located in sigma 70 domains involved in recognition of -10 and -35 DNA sequences, which are separated by ~17 base pairs. Thus, the predicted distance of 58 ± 6 Å is compatible with the distance expected from the ~17-base pair separation between binding sites for these two structural domains of the protein.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM50514 and American Cancer Society Grant RPG 94-010-03-NP.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.

Dagger To whom correspondence should be addressed: Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis University Medical School, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8152; Fax: 314-577-8156; E-mail: heydukt{at}wpogate.slu.edu.

The abbreviations used are: RNAP, RNA polymerase; ssDNA, single-stranded DNA; LRET, luminescence resonance energy transfer; AMCA-NHS, succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic acid; DTPA, diethylenetriaminepentaacetic acid; nt, nucleotide; DMF, N,N'-dimethylformamide.

2 In cases in which the donor decay contains an unquenched (no LRET) component, a very small amplitude decay component with a lifetime of unquenched donor is also observed in sensitized acceptor decay because of some residual emission of the donor at 670 nm, where the acceptor decay is observed.

3 S. Callaci, E. Heyduk, and T. Heyduk, unpublished data.

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Abstract
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