Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

A coiled-coil from the RNA polymerase beta' subunit allosterically induces selective nontemplate strand binding by sigma(70).

For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes.

Lanthanide chelates used as donors offer several advantages over classical fluorescence probes in resonance energy transfer distance measurements. One of these advantages is that energy transfer can be conveniently measured using sensitized acceptor decay measurements. In these measurements a long microsecond lifetime of the lanthanide donor and a short nanosecond lifetime of the acceptor allow elimination of a signal from the unquenched donor. Therefore, the decay of sensitized acceptor emission reflects decay properties of the donor engaged in energy transfer. The purpose of this work is to point out the importance of the fact that the amplitude of the sensitized acceptor signal is dependent on the resonance energy transfer rate constant. Thus, in the case where there are two or more populations of donors with different energy transfer rate constants, the relative amplitudes of corresponding decay components observed in sensitized acceptor emission do not represent the relative populations of the donors. We use simulations to show that these effects can be very significant. A minor population of donors with a high rate of energy transfer can produce sensitized acceptor decay which is dominated by a decay component corresponding to this minor donor population. Using a simple experimental system of rapid diffusion limit energy transfer between a europium chelate and Cy5 acceptor we show that the predicted dependency of sensitized acceptor decay amplitude on the energy transfer rate is indeed observed. We suggest that the relative importance of decay components observed in sensitized acceptor emission should be evaluated after an appropriate correction of their values such that they properly reflect possible different populations of donors. We describe a method to perform such correction.

HMG1 proteins from evolutionary distant organisms distort B-DNA conformation in similar way.

The abundant high-mobility group proteins 1/2 (HMG1/2) represent a group of potent architectural elements of chromatin. They are able to induce strong bends and untwist DNA. Here, we compared the abilities of diverse HMG1 proteins to distort the B-DNA conformation of 30-base pair DNA fragment. The DNA bending was measured in solution by monitoring fluorescence resonance energy transfer between fluorescence probes attached to opposite ends of the DNA fragment. Various insect and plant proteins which differ in size, in composition of their HMG1-box domains (HMG1-BD), and in composition of the N- and the C-terminally flanking regions were analyzed in these experiments. Despite these structural differences the extent of the induced changes in DNA conformation upon binding to various proteins was similar, as the estimated bend angle was 150+/-20 degrees for all the tested proteins. Our results suggest that a set of highly conserved residues stabilizing the tertiary structure of the HMG1-BD mainly determines the extent of DNA bending in the complex. Even extended positively charged regions flanking the HMG1-BD are apparently not able to influence this conformational distortion of DNA.

Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit.

Luminescence resonance energy transfer measurements were used to show that binding of E. coli core RNA polymerase induced major changes in interdomain distances in the sigma 70 subunit. The simplest model describing core-induced changes in sigma 70 involves a movement of the conserved region 1 by approximately 20 A and the conserved region 4.2 by approximately 15 A with respect to conserved region 2. The core-induced movement of region 1 (autoinhibition domain) and region 4.2 (DNA-binding domain) provides structural rationale for allosteric regulation of sigma 70 DNA binding properties by the core and suggests that this regulation may not only involve directly the autoinhibition domain of sigma 70 but also could involve a modulation of spacing between DNA-binding domains of sigma 70 induced by binding of core RNAP.

Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study.

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (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 sigma70 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 sigma70: 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 sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.

Conformational changes of Escherichia coli RNA polymerase sigma70 factor induced by binding to the core enzyme.

Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70. Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains.

Conformational changes of DNA induced by binding of Chironomus high mobility group protein 1a (cHMG1a). Regions flanking an HMG1 box domain do not influence the bend angle of the DNA.

High mobility group (HMG) proteins are thought to facilitate assembly of higher order chromatin structure through modulation of DNA conformation. In this work we investigate the bending of a 30-base pair DNA fragment induced by Chironomus HMG1 (cHMG1a), and HMGI (cHMGI) proteins. The DNA bending was measured in solution by monitoring the end-to-end distance between fluorescence probes attached to opposite ends of the DNA fragment. The distance was measured by fluorescence energy transfer using a novel europium chelate as a fluorescence donor. These measurements revealed that the end-to-end distance in the 30-base pair DNA was decreased from approximately 100 A in free DNA to approximately 50.5 A in cHMG1a. DNA complex. The most probable DNA bending angle consistent with these distance measurements is about 150 degrees. The deletion of the charged regulatory domains located close to the C terminus of the HMG1 box domain of cHMG1a protein had no effect on the induced bend angle. The ability to induce a large DNA bend distinguishes the cHMG1 from the cHMGI protein. Only small perturbation of the DNA conformation was observed upon binding of the cHMGI protein. A strong DNA bending activity of cHMG1a and its relative abundance in the cell suggests that this protein plays a very important role in modulation of chromatin structure.

Determinants for Escherichia coli RNA polymerase assembly within the beta subunit.

We used binding assays and other approaches to identify fragments of the Escherichia coli RNAP beta subunit involved in the obligatory interaction with the alpha subunit to form the stable assembly intermediate alpha2beta as well as in the interaction to recruit the beta' subunit into the alpha2beta sub-assembly. We show that two regions of evolutionarily conserved sequence near the C terminus of beta (conserved regions H and I) are central to the assembly of RNAP and likely make subunit-subunit contacts with both alpha and beta'.

Thiol-reactive, luminescent Europium chelates: luminescence probes for resonance energy transfer distance measurements in biomolecules.

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T. M. Rana, and J. E. Hearst (1994) J. Am. Chem. Soc. 116, 6029-6030; P. R. Selvin, and J. E. Hearst (1994) Proc. Natl. Acad. Sci. USA 91, 10024-10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of the R0 value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach.

A novel direct approach, analogous to DNA footprinting, for mapping protein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions with the allosteric ligand (cAMP) and DNA. In this approach, a protein-macromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA. The cleavage products are resolved by SDS-PAGE and transferred to a PVDF membrane. Transferred polypeptides are visualized by immunostaining with antibodies specific to the N-terminal peptide of the protein. The mobility of the bands visualized in such a way is directly proportional to the distance of the cleavage sites from the N-terminus, and thus the positions of the sites protected from cleavage by a bound macromolecule can be determined. Thus, protein domains involved in macromolecular interactions can be mapped. In the case of CRP, the cleavage conditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity. When applied to CRP-DNA interactions, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA. The obtained results showed also that the binding of CRP to the DNA binding site perturbed the region of CRP involved in intersubunit interactions. An allosteric ligand (cAMP) appeared to perturb the same region of CRP. This stresses out the importance of intersubunit interactions in cAMP modulation of protein DNA binding affinity. The protein footprinting methodology presented in this paper should be broadly generalizable to any protein-macromolecule system.

Electrostatic forces contribute to interactions between trp repressor dimers.

The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers.

Physical studies on interaction of transcription activator and RNA-polymerase: fluorescent derivatives of CRP and RNA polymerase.

Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe the preparation of fluorescent derivatives of CRP (fluorescent probes at position 13 and 85); and of the alpha-subunit of RNAP (at position 321). The specific incorporation of fluorescence probes was achieved by expressing protein in a bacteria strain, auxotrophic for tryptophan, in media containing 5-hydroxytryptophan (5-OH-Trp). The absorbance spectrum of a protein containing 5-OH-Trp is shifted towards longer wavelengths as compared to the native protein. This allows selective monitoring of the fluorescence signal of 5-OH-Trp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to retain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown that 5-OH-Trp derivative of CRP interacts with RNAP as well as the native protein. The RNAP reconstituted with 5-OH-Trp derivative of the alpha-subunit retained the enzymatic activity. Fluorescence quenching studies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain.

Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Global conformational changes in allosteric proteins. A study of Escherichia coli cAMP receptor protein and muscle pyruvate kinase.

One of the basic features in allosteric regulation involves long range transduction of information. Based on crystallographic data on protein systems that are regulated by allosteric mechanisms, a global conformational change has always been observed. It is, therefore, important and useful to correlate the cooperativity of global structural change with the mode of binding of the regulatory ligand. Two systems were chosen for study, namely Escherichia coli cAMP receptor protein and muscle pyruvate kinase, which show negative and positive cooperativity in the binding of allosteric ligands, respectively. Quantitative titration of the global structural change, monitored by a high precision analytical gel chromatography technique, was conducted as a function of allosteric effector concentration. The results obtained for cAMP receptor protein show that the protein undergoes contraction upon binding of cAMP. The decreases in Stokes radius associated with complex formation are 0.1 +/- 0.1 and 0.7 +/- 0.1 A when one and two cAMP-binding sites are filled, respectively. The results for the pyruvate kinase system show a concerted structural change that quantitatively match the predicted behavior based on equilibrium constants derived from the analysis of steady state kinetic data by a two-state model. Hence, for these two systems, these results show that negative and positive cooperativity are correlated with sequential and concerted modes of structural change, respectively.