Marked stepwise differences within a common kinetic mechanism characterize TATA-binding protein interactions with two consensus promoters.

Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA sequence is an obligatory initial step in RNA polymerase II transcription initiation. The interactions of Saccharomyces cerevisiae TBP with the E4 (TATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled via global analysis of kinetic and thermodynamic data obtained using fluorescence resonance energy transfer. A linear two-intermediate kinetic mechanism describes the reaction of both of these consensus strong promoters with TBP. Qualitative features common to both interactions include tightly bound TBP-DNA complexes with similar solution geometries, simultaneous DNA binding and bending, and the presence of intermediate TBP-DNA conformers at high mole fraction throughout most of the reaction and at equilibrium. Despite very similar energetic changes overall, the stepwise entropic and enthalpic compensations along the two pathways differ markedly following the initial binding/bending event. Furthermore, TBP-E4 dissociation ensues from both replacement and displacement processes, in contrast to replacement alone for TBP-adenovirus major late promoter. A model is proposed that explicitly correlates these similarities and differences with the sequence-specific structural properties inherent to each promoter. This detailed mechanistic comparison of two strong promoters interacting with TBP provides a foundation for subsequent comparison between consensus and variant promoter sequences reacting with TBP.

DNA sequence-dependent differences in TATA-binding protein-induced DNA bending in solution are highly sensitive to osmolytes.

The complex formed between the TATA-binding protein (TBP) and the "TATA box" of eukaryotic class II promoters is the foundation for assembly of the complex to which RNA polymerase II is ultimately recruited. TBP binds productively to canonical and diverse variant TATA sequences with >100-fold differences in transcription efficiency. Co-crystals of canonical sequences and >11 variant sequences bound to various TBP molecules all have approximately 80 degrees bends. In contrast, the bend angles for TBP-TATA complexes in solution, derived from distance distributions, are approximately 80 degrees for a canonical sequence but range from 30 degrees to 62 degrees for five variant sequences. We show in this study that the osmolytes used to crystallize TBP-TATA complexes induce profound increases in the DNA bends of two transcriptionally active TBP-bound variant sequences to a common angle of approximately 80 degrees but have little effect on a transcriptionally inactive variant. The effect of osmolyte on the TBP-induced DNA bend of a variant TATA box sequence is also manifest in the kinetics of association, demonstrating a functional consequence of an osmolyte-induced structural change.

DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequence-dependent.

The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences. The sequence dependence of the DNA structure in TBP-TATA complexes has been investigated in solution using fluorescence resonance energy transfer. The mean 5'dye-3'dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76 degrees and for the variants ranging from 30 degrees to 62 degrees. These solution bends contrast sharply with the corresponding co-crystal structures, which show approximately 80 degrees bends for all sequences. Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP-DNA binding affinities. Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP-TATA binary complex. Specifically, the distance distribution for the average solution structure of the TBP-TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent. Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.

Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes.

Time-resolved Förster resonance energy transfer (trFRET) has been used to obtain interdye distance distributions. These distributions give the most probable distance as well as a parameter, sigma, that characterize the width of the distribution. This latter parameter contains information not only on the flexibility of the dyes tethered to macromolecules, but on the flexibility of the macromolecules. Both the most probable interdye distance as well as sigma provide insight into DNA static bending and DNA flexibility. Time-resolved fluorescence anisotropy and static anisotropy measurements can be combined to provide a measure of the cone angle within which the tethered dyes appear to wobble. When this motion is an order of magnitude faster than the average lifetime that characterizes transfer, an average value of the dipolar orientational parameter kappa2 can be calculated for various mutual dye orientations. The resulting kappa2 distribution is very much narrower than the limiting values of 0 and 4, allowing more precise distances and distance changes to be determined. Static and time-resolved fluorescence data can be combined to constrain the analyses of DNA-protein kinetics to provide thermodynamic parameters for binding and for conformational changes along a reaction coordinate. The parameter sigma can be used to model multiple DNA-protein complexes with varying DNA bend angles in a global fitting of trFRET data. Such a global fitting approach has shown how the range of bends in single base DNA variants, when bound by the TATA binding protein (TBP), can be understood in terms of two limiting forms. Time-resolved FRET, combined with steady-state FRET, can be used to show not only how osmolytes affect the binding of DNA to proteins, but also how DNA bending depends on osmolyte concentration in the DNA-protein complexes.

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex.

Binding of the TATA-binding protein (TBP) to the "TATA" sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements.

The binding and bending of tetramethylrhodamine-5'-(GGGCTATAAAAGGG) duplex-3'-fluorescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer. Probability distributions derived from fluorescein emission lifetime measurements show a decrease in the mean 3'-fluorescein-5'- rhodamine distance from 56.5 to 46.8 A upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography. The kinetics, monitored in real time using stopped flow fluorimetry, demonstrate simultaneous binding and bending of a TATA box by TBP with a single second-order rate constant of (2.4 +/- 0.3) x 10(6) M-1 s-1 at 30 degrees C.

Kinetic studies by fluorescence resonance energy transfer employing a double-labeled oligonucleotide: hybridization to the oligonucleotide complement and to single-stranded DNA.

A single 16-base oligodeoxyribonucleotide was labeled at the 3'-end with fluorescein and at the 5'-end with x-rhodamine (R*oligo*F); the chromophores served as a donor/acceptor pair, respectively, for Förster resonance energy transfer. We exploited the striking differences in the steady-state emission spectra of the R*oligo*F as a single strand and in a duplex structure to signal hybridization in solution and to determine the kinetics of duplex formation as the probe bound to its oligomer complement and to its target sequence in M13mp18(+) phage DNA. The binding followed second-order kinetics; in 0.18 M NaCl (pH 8) with 25% formamide, the rate constant for binding to the oligomer complement was 5.7 x 10(5) M-1 s-1, and that to M13mp18(+) was 5.7 x 10(4) M-1 s-1. The source of the 10-fold decrease in the rate of binding to M13mp18(+) was examined to differentiate between multiple nonproductive nucleation and rapid fluctuations in the structure around the target site. From simulations based on each model combined with associated experimental results, we concluded that the slower binding was due to rapid structural fluctuations around the target site, with an effective target concentration 0.1 of that of the total. Comparisons of total fluorescein emission derived from both steady-state and lifetime measurements suggest that the 5'-x-rhodamine induces a conformational change that affects the interaction at the 3'-end between the fluorescein and the polymer. The effects of salt on the fluorescence were complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Donor-acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes.

A 16-mer deoxyribonucleotide was labeled at the 5'-end with x-rhodamine and at the 3'-end with fluorescein. Förster resonance energy transfer was used to determine the distribution, P(R), of donor-acceptor distances for the oligomer in three duplex structures (hybridized to complementary strands having 10, 16, and 24 bases) and as a single strand; measurements were made in 0.18 M NaCl and 1 M KCl solutions. These distributions were derived from lifetime measurements made in the frequency domain using a multiharmonic frequency phase fluorometer. The fluorescein fluorescence decay for each duplex structure was fit very well with P(R) modeled as a shifted Gaussian. On the basis of the mean donor-acceptor distance, these structures for the 16-mer and the 24-mer were consistent with that of B-DNA. For the 16-mer duplex in 0.18 M NaCl, the distribution was centered at 68.4 A with sigma = 6.4 A. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 A, is much larger than that for a structure with the fluorophores extended perpendicular to the helix axis. For the single-strand data, various high-quality fits yielded physically unreasonable distributions or could not accurately account for the acceptor response. Considered analyses suggested that the single-strand distribution was best represented by a shifted Gaussian, with R = 51.5 A and sigma = 10.0 A in 0.18 M NaCl. In all cases, sigma increased and R decreased in 1 M KCl relative to their values in 0.18 M NaCl, consistent with the increased flexibility of the polymer.

Thermodynamic characterization of the cooperativity of 40S complex formation during the initiation of eukaryotic protein synthesis.

The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.

Synthesis and characterization of conjugates formed between periodate-oxidized ribonucleotides and amine-containing fluorophores.

The synthesis and purification of new fluorescently labeled derivatives of GDP and ATP are described. The fluorescent groups are coupled initially through amine-containing linker arms to periodate-oxidized nucleotides. Reduction of the initial product yields primarily a six-membered morpholine-like ring. Fluorescein-labeled GDP, rhodamine-labeled GDP, and fluorescein-labeled ATP were characterized by absorbance spectroscopy and TLC. NMR and FAB-MS studies were carried out on a single nucleotide derivative formed by reacting periodate-oxidized guanosine and benzylamine with subsequent reduction to establish the modification to the ribose moiety. The synthesis of the guanosine-benzylamine conjugate led to a mixture of products that were separated. The predominant product (70%) resulted in conversion of the ribose moiety to a six-membered morpholine-like ring having no hydroxyl group, and the minor product (30%) resulted in an open ring structure having one hydroxyl. When NaCNBH3 was used as the sole reductant, only the product with the morpholine-like ring was formed. These probes were prepared for use in solution studies of the interactions of eukaryotic initiation factor-2 with other components of mammalian protein synthesis initiation.

Synthesis of dansyl ribonucleotides and their use in steady-state fluorescence anisotropy studies of nucleotide binding by initiation factor-2 (eIF-2) and histone H1.

1. Fluorescent analogs of GDP and ATP were prepared with DANSYL-beta-alanine (D beta A) coupled to the (2')3' hydroxyl of the ribose. 2. Observation of changes in both total fluorescence and anisotropy accompanying the binding of D beta A-GDP to eIF-2 allowed determination of Kd (33 nM). 3. When D beta A-ATP bound to H1 histone, the fluorescence quantum yield increased and the emission was blue shifted. Analysis yielded a Kd of 3.4 microM and 20 binding sites per histone. At high levels of ATP, fluorescence anisotropy values and light scattering intensities pointed to significant aggregation of H1 that is strongly dependent on ATP concentration.

A pulsed photolysis procedure for determining oxygen equilibrium parameters of low-affinity noncooperative hemoglobins.

It is often very difficult to obtain precise values for oxygen equilibrium constants for low-affinity noncooperative hemoglobins owing to the ease with which they are oxidized and denatured with even the most gentle stirring or agitation. We have developed a procedure that eliminates this problem by maintaining the hemoglobin as HbCO except during the brief time it is present as Hb, and HbO2 forms following photolysis. Oxygen is continuously removed by an enzyme system. The solution is photolyzed to 100% every 2-3 min during the deoxygenation process to obtain maximum absorbance changes at the Hb-HbCO isosbestic. These maximum absorbance changes at known times during the deoxygenation provide the necessary data for obtaining the oxygen equilibrium constant. These absorbance changes are used with enzyme kinetics equations to obtain calculated times. The simple equations, which neglect heme concentration, will be satisfactory for nearly all conditions, but for generality the complete equations are given. The variance minimizations are with respect to observed and calculated photolysis times. The results for native carp Hb over a range of temperatures are in excellent agreement with results obtained with considerably more material and greater difficulty by other methods.

Rapid preparation of native alpha and beta chains of human hemoglobin.

1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is less than 0.1%. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins.

1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid autoxidation. 2. In carp Hb (pH 6 + IHP, 25 degrees C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P50 or X. 3. We circumvent this problem by rapidly flowing HbO2, initially at pH 9 (X = 1.4 microM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained less than 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O2 activity. 4. At 22 degrees C, the half-saturation oxygen activity (X) is 227 microM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O2 affinities of the alpha and beta chain hemes or, two different T states.

Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin.

A small stopped-flow cuvette was built into a computer-controlled Cary 210 spectrophotometer. The enzymatic depletion of oxygen in solutions of hemoglobin and myoglobin was initiated by flowing the hemeproteins with the enzyme against a solution of the hemeproteins containing the appropriate substrate. The deoxygenation was homogeneous throughout the solution. Oxygen activity was calculated at each instant of time from the fractional saturation of Mb, determined from observations at the Hb/HbO2 isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO2 isosbestic wavelength. The spectrophotometer cycled between these two wavelengths during the deoxygenation. The deoxygenation of HbO2 was largely complete in 20-25 min, whereas the deoxygenation of MbO2 was allowed to proceed for about 1 h. This procedure eliminates equilibration of Hb solutions with a gas phase and replaces oxygen electrode readings with spectrophotometric sensing by Mb, providing essentially instantaneous determinations of oxygen activity and hence 250-500 or more independent data points per run. The Mb and Hb data vectors require several manipulations to correct for small relative displacements in time and for small non-isosbestic effects. Detailed consideration of the enzyme kinetics allowed oxygen activities to be determined in regions where Mb is a poor sensor. Studies of HbO2 deoxygenation as a function of wavelength show that the determination of the four Adair constants requires in addition the determination of three spectroscopic parameters. Values of the apparent Adair constants, determined without these spectroscopic parameters, depend strongly on the monitoring wavelength.