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M Brenowitz

Publications and source records attributed to M Brenowitz.

At least 19 recordsLinked to original sources
PubMed2001

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis.

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.

ATP-Dependent Proteases↗
PubMed2001

Kinetic trapping of DNA by transcription factor IIIB.

High levels of RNA polymerase III gene transcription are achieved by facilitated recycling of the polymerase on transcription factor IIIB (TFIIIB)-DNA complexes that are stable through multiple rounds of initiation. TFIIIB-DNA complexes in yeast comprise the TATA-binding protein (TBP), the TFIIB-related factor TFIIIB70, and TFIIIB90. The high stability of the TFIIIB-DNA complex is conferred by TFIIIB90 binding to TFIIIB70-TBP-DNA complexes. This stability is thought to result from compound bends introduced in the DNA by TBP and TFIIIB90 and by protein-protein interactions that obstruct DNA dissociation. Here we present biochemical evidence that the high stability of TFIIIB-DNA complexes results from kinetic trapping of the DNA. Thermodynamic analysis shows that the free energies of formation of TFIIIB70-TBP-DNA (DeltaG degrees = -12.10 +/- 0.12 kcal/mol) and TFIIIB-DNA (DeltaG degrees = -11.90 +/- 0.14 kcal/mol) complexes are equivalent whereas a kinetic analysis shows that the half-lives of these complexes (46 +/- 3 min and 95 +/- 6 min, respectively) differ significantly. The differential stability of these isoenergetic complexes demonstrates that TFIIIB90 binding energy is used to drive conformational changes and increase the barrier to complex dissociation.

DNA↗
PubMed2001

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.

Adenovirus E4 Proteins↗
PubMed2001

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.

Base Sequence↗
PubMed2000

A detailed interpretation of OH radical footprints in a TBP-DNA complex reveals the role of dynamics in the mechanism of sequence-specific binding.

The hydroxyl radical footprint of the TATA-binding protein (TBP) bound to the high-affinity sequence TATAAAAG of the adenovirus 2 major late promoter has been quantitatively compared to a 2 ns molecular dynamics simulation of the complex in aqueous solution at room temperature using the CHARMM23 potential. The nucleotide-by-nucleotide analysis of the TBP-TATA hydroxyl radical footprint correlates with the solvent-accessible surface calculated from the dynamics simulation. The results suggest that local reactivity towards OH radicals results from the interplay between the local DNA geometry imposed by TBP binding, and the dynamics of the side-chains contacting the sugar hydrogen atoms. Analysis of the dynamics suggests that, over time, TBP forms stable interactions with the sugar-phosphate backbone through multiple contacts to different partners. This mechanism results in an enthalpic advantage to complex formation at a low entropic cost.

Adenine↗
PubMed2000

Folding mechanism of the Tetrahymena ribozyme P4-P6 domain.

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozyme, which forms a stable, closely packed tertiary structure. The 160-nt domain folds independently at a similar rate (approximately 2 s(-1)) as it does in the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl(2). Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k >/= 50 s(-1)) in isolation, than when part of the wild-type P4-P6 RNA. This difference implies that long-range interactions in the P4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Analytical centrifugation was used to measure the sedimentation and diffusion coefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreased from 58 to 46 A over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg(2+) causes the domain to collapse to a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conformation of the RNA in higher salt conditions.

Animals↗
PubMed2000

Participation of the amino-terminal domain in the self-association of the full-length yeast TATA binding protein.

The association of monomeric TATA binding protein with promoter DNA is an essential first step in many current models of eukaryotic transcription initiation. This step is followed by others in which additional transcription factors, and finally RNA polymerase, assemble at the promoter. Here we characterize the quaternary interactions of the Saccharomyces cerevisiae TATA-binding protein (yTBP), in the absence of other proteins or DNA. The data reveal a robust pattern in which yTBP monomers equilibrate with tetramers and octamers over a broad span of temperatures (4 degrees C </= T </= 37 degrees C) and salt concentrations (60 mM </= [KCl] </= 1 M), that includes the physiological range. Association is highly cooperative, with octamer formation favored by approximately 9 kcal/mol over tetramer formation. Changes in association constant with [KCl] are consistent with an assembly-linked release of ions at low salt and an assembly-linked uptake of ions at high salt, for both monomer right arrow over left arrow tetramer and tetramer right arrow over left arrow octamer reaction steps. Fluorescence emission spectra and steady-state anisotropies reveal that the amino-terminal domain changes conformation and dynamics at both association steps and that the polarity of the environment near tryptophan 26 is sensitive to changes in [KCl] in the monomeric and tetrameric states but not the octameric state. These results are consistent with a [salt]-dependent change in the assembly mechanism near 300 mM KCl and suggest that the amino-terminal domain may modulate the self-association of the full-length protein. TBP self-association may regulate many of its cellular functions, including transit of the nuclear membrane and participation in transcription initiation.

Allosteric Regulation↗
PubMed2000

Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation.

For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods. We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme. The cooperativity of folding, m-value, and the free energy, DeltaG degrees N-U, associated with formation of each tertiary contact was determined by analysis of the isotherms. The DeltaG degrees N-U values measured in this study vary from 1.7 +/- 0.2 to 7. 6 +/- 1.2 kcal mol-1. Thus, the stability of these discrete tertiary contacts vary by almost 104. In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol-1 M-1) indicating that these contacts exhibit global cooperatively in their folding behavior. This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.

Allosteric Site↗
PubMed1999

Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry.

Radiolysis of peptide and protein solutions with high-energy X-ray beams induces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radiolysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniques were used to characterize radiolytic modification to amino acids cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity order of Cys, Met >> Phe, Tyr, > Trp > Pro > His, Leu was determined under aerobic reaction conditions from MS/MS analysis of the radiolyzed peptide products. Radiolysis of peptides in 18O-labeled water under aerobic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine and methionine side chains reacted with hydroxyl radicals generated from radiolysis of water as well as molecular oxygen. Phenylalanine and tyrosine residues were modified predominantly by hydroxyl radicals, and the source of modification of proline was exclusively through molecular oxygen.

Amino Acid Sequence↗
PubMed1999

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.

Adenoviridae↗
PubMed1999

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers.

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.

DNA-Binding Proteins↗
PubMed1998

DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding.

The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables.

Base Sequence↗
PubMed1998

RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.

Radiolysis of water with a synchrotron x-ray beam permits the hydroxyl radical-accessible surface of an RNA to be mapped with nucleotide resolution in 10 milliseconds. Application of this method to folding of the Tetrahymena ribozyme revealed that the most stable domain of the tertiary structure, P4-P6, formed cooperatively within 3 seconds. Exterior helices became protected from hydroxyl radicals in 10 seconds, whereas the catalytic center required minutes to be completely folded. The results show that rapid collapse to a partially disordered state is followed by a slow search for the active structure.

Animals↗
PubMed1998

DNA structure and flexibility in the sequence-specific binding of papillomavirus E2 proteins.

The papillomavirus E2 proteins are transcriptional regulators that bind to a consensus DNA sequence ACCG NNNN CGGT. Multiple copies of this binding site are found in the viral genomes. The affinities of the naturally occurring binding sites for the E2 proteins are predominantly dependent upon the sequence of the NNNN spacer. The hierarchies of binding site affinities among the sites present in the viral genomes result in differential occupancy during the viral life-cycle. In turn, this differential binding regulates transcription from viral promoters, including those for the oncogenes E6 and E7. Structural and biochemical studies have shown that E2 proteins bend the DNA to which they specifically bind. Atomic resolution structures of complexes of the bovine papillomavirus strain 1 (BPV-1) E2 protein and DNA show that the protein does not contact the spacer DNA. A direct comparison of the binding of the DNA-binding domains of the E2 proteins from BPV-1 and human papillomavirus strain 16 (HPV-16) to a series of binding sites as a function of the sequence of their central spacer and/or the presence of a nick or gap in one strand of the spacer DNA is presented in this paper. The BPV-1 E2 DNA-binding domain is only moderately sensitive to the nature of the central spacer; less than several fold differences in affinity were observed when the DNA sequence of the spacer was varied and/or a nick or gap was introduced. In contrast, the HPV-16 E2 DNA-binding domain binds to sites containing A:T-rich central spacers with significantly increased affinity. The introduction of a nick or gap into the spacer of these high affinity sequences is very detrimental to HPV-16 E2 binding while comparable nicks or gaps have only small effects in the low affinity sequences. These results suggest that the HPV-16 E2 protein recognizes the structure of the DNA spacer and that the mechanism of DNA-sequence specific binding of the homologous HPV-16 E2 and BPV-1 E2 proteins is significantly different.

Animals↗
PubMed1998

The TATA element and its context affect the cooperative interaction of TATA-binding protein with the TFIIB-related factor, TFIIIB70.

We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATA-flanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB70. TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding. We also determined the cooperative free energies of formation of TBP.TFIIIB70.DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element. The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP. In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12- and 17-fold, respectively. Since the TBP-TFIIIB70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes. An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB70 cooperativity. The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.

Adenoviridae↗
PubMed1998

Following the folding of RNA with time-resolved synchrotron X-ray footprinting.

The rapid mixing synchrotron X-ray footprinting technique described in this article allows nucleic acid folding and ligand binding reactions to be followed on a millisecond time resolution with single nucleotide resolution. In principle, the change in .OH protection of every nucleotide in a nucleic acid hundreds of nucleotides long can be monitored separately. In addition, a wide range of solution conditions are compatible with the radiolytic generation of .OH. These characteristics of synchrotron X-ray footprinting create opportunities for conducting thermodynamic and kinetic studies of nucleic acids that are both comprehensive and detailed. Kinetic footprinting studies of a number of systems have been initiated by the Center for Synchrotron Biosciences using this technique.

Flow Injection Analysis↗
PubMed1997

Comparison of the DNA association kinetics of the Lac repressor tetramer, its dimeric mutant LacIadi, and the native dimeric Gal repressor.

The rates of association of the tetrameric Lac repressor (LacI), dimeric LacIadi (a deletion mutant of LacI), and the native dimeric Gal repressor (GalR) to DNA restriction fragments containing a single specific site were investigated using a quench-flow DNase I "footprinting" technique. The dimeric proteins, LacIadi and GalR, and tetrameric LacI possess one and two DNA binding sites, respectively. The nanomolar protein concentrations used in these studies ensured that the state of oligomerization of each protein was predominantly either dimeric or tetrameric, respectively. The bimolecular association rate constants (ka) determined for the LacI tetramer exceed those of the dimeric proteins. The values of ka obtained for LacI, LacIadi, and GalR display different dependences on [KCl]. For LacIadi and GalR, they diminish as [KCl] increases from 25 mM to 200 mM, approaching rates predicted for three-dimensional diffusion. In contrast, the ka values determined for the tetrameric LacI remain constant up to 300 mM [KCl], the highest salt concentration that could be investigated by quench-flow footprinting. The enhanced rate of association of the tetramer relative to the dimeric proteins can be modeled by enhanced "sliding" (Berg, O. G., Winter, R. B., and von Hippel, P. H. (1981) Biochemistry 20, 6929-6948) of the LacI tetramer relative to the LacIadi dimer or a combination of enhanced sliding and the superimposition of "direct transfer" mediated by the bidentate DNA interactions of the tetramer.

Bacterial Proteins↗