From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104
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ABSTRACT |
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We used luminescence energy transfer measurements
to determine the localization of 5'- and 3'-ends of a 12-nucleotide
nontemplate strand oligonucleotide bound to 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.
70
holoenzyme. Five single reactive cysteine mutants of
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
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
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
70 is located in an
-helix containing
residue 442. The 5'-end of the oligonucleotide was oriented toward the
COOH terminus of the helix.
INTRODUCTION
Top
Abstract
Introduction
References
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.
(Eq. 1)
The E. coli RNA polymerase holoenzyme is a multisubunit
enzyme (subunit composition 2
'
) (1).
70 (the primary
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
70
and eukaryotic single-stranded DNA (ssDNA)-binding proteins was used as
a basis of the proposal that
70 subunit could also be
actively involved in the promoter melting reaction through binding of
ssDNA of the open complex (7). This favorable ssDNA-
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
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
70 is most likely located in the conserved region 2.3 of the
70 subunit (13, 14, 16, 19-21). The ssDNA
binding activity of
70 is regulated allosterically
through binding to the core RNAP (16). Free
70 binds
ssDNA weakly and does not discriminate between the template and
nontemplate sequences. Binding of
70 to the core reduces
the affinity of
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
70 are consistent with its active
role in the promoter melting reaction.
Region 2.3 of 70 is thought to be a location of the
ssDNA binding site of
70. However, the architecture of
the ssDNA-
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
70 with
respect to several functional domains of the protein.
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EXPERIMENTAL PROCEDURES |
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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 70--
The
preparation of single-cysteine mutants of
70 is
described elsewhere (27). Briefly, three endogenous cysteine residues of
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]
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
70 were used in LRET experiments:
[1C]
70, [A59C]
70,
[S366C]
70, [S442C]
70, and
[R596C]
70. Single-cysteine mutants of
70 were expressed and purified as described before
(27). Transcriptional activity of single-cysteine mutants of
70 compared with the wild type
70 was:
109% ([1C]
70), 83% ([A59C]
70), 64%
([S366C]
70), 95% ([S442C]
70), and
100% ([R596C]
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 70--
Samples of
each single-cysteine mutant of
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
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
70 on a fast protein
liquid chromatography Superdex-200 size exclusion column (Amersham
Pharmacia Biotech). For experiments with free donor-labeled
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,
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
The energy transfer (E) between europium chelate-labeled
70 and Cy5-labeled oligonucleotide was calculated from
measurements of luminescence lifetime of a donor in the absence
(
d) and in the presence of acceptor
(
da).
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(Eq. 5) |
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(Eq. 6) |
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RESULTS |
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Acceptor-labeled 12-nt Oligonucleotide and Donor-labeled
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
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. 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
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
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
70 ([S366C]
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
70 proteins was
0.5-1.0 mol of AMCA-DTPA/mol of protein.
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LRET Measurements--
Figs. 3 and
4 show representative examples of LRET
data for the donor-labeled 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]
70 and 12-nt oligonucleotide
labeled with Cy5 at the 5'-end (Cy5-5'-NT). The results for other
donor-labeled
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
70 and labeled oligonucleotides. LRET data for all of
these combinations are summarized in Tables II and III.
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In the absence of acceptor-labeled oligonucleotide, luminescence decays
of donor-labeled 70 reconstituted with the core RNAP
were monoexponential (Fig. 3A). In the presence of
acceptor-labeled oligonucleotide, decays of donor-labeled
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
(
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
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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Two lifetimes (1 and
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]
70 and NT-3'-Cy5 DNA
these lifetimes were:
1 = 66 µs (9% of total fast
component amplitude) and
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
1 and
2 were very similar to distances
calculated using only
2 (the average difference in
distances between these two modes of calculation was only 4 ± 2 Å).
LRET between the donor-labeled 70 and acceptor-labeled
12-nt nontemplate strand oligonucleotide was specific only for
70 reconstituted with the core RNAP. Free
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
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
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 70-Nontemplate ssDNA
Complex--
Results of LRET measurements with all five
single-cysteine mutants of
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
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
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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Using distances determined by LRET, three-dimensional models of
relative localization of different domains of 70 could
be built. An example of such a model superimposed on the crystal
structure of
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'
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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DISCUSSION |
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We have determined the distances between several sites in the
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' 3' or 3'
5' orientation. If the binding could occur equally well in either orientation, the apparent distances measured between sites in
70 and the 5'- or
the 3'-end of the oligonucleotide should be the same. We observed,
however, that for several sites in
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
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
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
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
70 seems to be well suited for the tasks that the ssDNA
binding site of
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 70. This is consistent and confirms the
proposals that the ssDNA binding site of
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
-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'
COOH terminus of the helix. Such an orientation
is consistent with the proposed model of nontemplate
ssDNA-
70 interaction based on the crystal structure of
the
70 fragment (37). It is also consistent with data
relating mutations in
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'
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 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 70 fragment (37). The agreement of this
predicted distance with the crystal structure of the
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
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.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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