High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase.

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.

Quantifying Amide-Aromatic Interactions at Molecular and Atomic Levels: Experimentally Determined Enthalpic and Entropic Contributions to Interactions of Amide sp2O, N, C and sp3C Unified Atoms with Naphthalene sp2C Atoms in Water.

In addition to amide hydrogen bonds and the hydrophobic effect, interactions involving π-bonded sp2 atoms of amides, aromatics, and other groups occur in protein self-assembly processes including folding, oligomerization, and condensate formation. These interactions also occur in aqueous solutions of amide and aromatic compounds, where they can be quantified. Previous analysis of thermodynamic coefficients quantifying net-favorable interactions of amide compounds with other amides and aromatics revealed that interactions of amide sp2O with amide sp2N unified atoms (presumably C═O···H-N hydrogen bonds) and amide/aromatic sp2C (lone pair π, n-π*) are particularly favorable. Sp3C-sp3C (hydrophobic), sp3C-sp2C (hydrophobic, CH-π), sp2C-sp2C (hydrophobic, π-π), and sp3C-sp2N interactions are favorable, sp2C-sp2N interactions are neutral, while sp2O-sp2O and sp2N-sp2N self-interactions and sp2O-sp3C interactions are unfavorable. Here, from determinations of favorable effects of 14 amides on naphthalene solubility at 10, 25, and 45 °C, we dissect amide-aromatic interaction free energies into enthalpic and entropic contributions and find these vary systematically with amide composition. Analysis of these results yields enthalpic and entropic contributions to intrinsic strengths of interactions of amide sp2O, sp2N, sp2C, and sp3C unified atoms with aromatic sp2C atoms. For each interaction, enthalpic and entropic contributions have the same sign and are much larger in magnitude than the interaction free energy itself. The amide sp2O-aromatic sp2C interaction is enthalpy-driven and entropically unfavorable, consistent with direct chemical interaction (e.g., lone pair-π), while amide sp3C- and sp2C-aromatic sp2C interactions are entropy-driven and enthalpically unfavorable, consistent with hydrophobic effects. These findings are relevant for interactions involving π-bonded sp2 atoms in protein processes.

Quantifying Amide-Aromatic Interactions at Molecular and Atomic Levels: Experimentally-determined Enthalpic and Entropic Contributions to Interactions of Amide sp 2 O, N, C and sp 3 C Unified Atoms with Naphthalene sp 2 C Atoms in Water.

In addition to amide hydrogen bonds and the hydrophobic effect, interactions involving π-bonded sp 2 atoms of amides, aromatics and other groups occur in protein self-assembly processes including folding, oligomerization and condensate formation. These interactions also occur in aqueous solutions of amide and aromatic compounds, where they can be quantified. Previous analysis of thermodynamic coefficients quantifying net-favorable interactions of amide compounds with other amides and aromatics revealed that interactions of amide sp 2 O with amide sp 2 N unified atoms (presumably C=O···H-N hydrogen bonds) and amide/aromatic sp 2 C (lone pair-π, n-π * ) are particularly favorable. Sp 3 C-sp 3 C (hydrophobic), sp 3 C-sp 2 C (hydrophobic, CH-π), sp 2 C-sp 2 C (hydrophobic, π-π) and sp 3 C-sp 2 N interactions are favorable, sp 2 C-sp 2 N interactions are neutral, while sp 2 O-sp 2 O and sp 2 N-sp 2 N self-interactions and sp 2 O-sp 3 C interactions are unfavorable. Here, from determinations of favorable effects of fourteen amides on naphthalene solubility at 10, 25 and 45 °C, we dissect amide-aromatic interaction free energies into enthalpic and entropic contributions and find these vary systematically with amide composition. Analysis of these results yields enthalpic and entropic contributions to intrinsic strengths of interactions of amide sp 2 O, sp 2 N, sp 2 C and sp 3 C unified atoms with aromatic sp 2 C atoms. For each interaction, enthalpic and entropic contributions have the same sign and are much larger in magnitude than the interaction free energy itself. The amide sp 2 O-aromatic sp 2 C interaction is enthalpy-driven and entropically unfavorable, consistent with direct chemical interaction (e.g. lone pair-π) while amide sp 3 C- and sp 2 C-aromatic sp 2 C interactions are entropy-driven and enthalpically unfavorable, consistent with hydrophobic effects. These findings are relevant for interactions involving π-bonded sp 2 atoms in protein processes.

High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for Mycobacterium tuberculosis RNA polymerase.

The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α- 32 P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription. Significance Statement: RNA polymerase transcription mechanisms have largely been determined from in vitro kinetic and structural biology methods. In contrast to the limited throughput of these approaches, in vivo RNA sequencing provides genome-wide measurements but lacks the ability to dissect direct biochemical from indirect genetic mechanisms. Here, we present a method that bridges this gap, permitting high-throughput fluorescence-based measurements of in vitro steady-state transcription kinetics. We illustrate how an RNA-aptamer-based detection system can be used to generate quantitative information on direct mechanisms of transcriptional regulation and discuss the far-reaching implications for future applications.

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein.

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

Experimentally determined strengths of favorable and unfavorable interactions of amide atoms involved in protein self-assembly in water.

Folding and other protein self-assembly processes are driven by favorable interactions between O, N, and C unified atoms of the polypeptide backbone and side chains. These processes are perturbed by solutes that interact with these atoms differently than water does. Amide NH···O=C hydrogen bonding and various π-system interactions have been better characterized structurally or by simulations than experimentally in water, and unfavorable interactions are relatively uncharacterized. To address this situation, we previously quantified interactions of alkyl ureas with amide and aromatic compounds, relative to interactions with water. Analysis yielded strengths of interaction of each alkylurea with unit areas of different hybridization states of unified O, N, and C atoms of amide and aromatic compounds. Here, by osmometry, we quantify interactions of 10 pairs of amides selected to complete this dataset. An analysis yields intrinsic strengths of six favorable and four unfavorable atom-atom interactions, expressed per unit area of each atom and relative to interactions with water. The most favorable interactions are sp2O-sp2C (lone pair-π, presumably n-π*), sp2C-sp2C (π-π and/or hydrophobic), sp2O-sp2N (hydrogen bonding) and sp3C-sp2C (CH-π and/or hydrophobic). Interactions of sp3C with itself (hydrophobic) and with sp2N are modestly favorable, while sp2N interactions with sp2N and with amide/aromatic sp2C are modestly unfavorable. Amide sp2O-sp2O interactions and sp2O-sp3C interactions are more unfavorable, indicating the preference of amide sp2O to interact with water. These intrinsic interaction strengths are used to predict interactions of amides with proteins and chemical effects of amides (including urea, N-ethylpyrrolidone [NEP], and polyvinylpyrrolidone [PVP]) on protein stability.

Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by Escherichia coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex.

FRET (fluorescence resonance energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3-Cy5 distance to ∼75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper ß'-clamp while downstream DNA contacts the top of the ß-pincer in I1E. Converting I1E to I1M (∼1 s time scale) reduces FRET efficiency with little change in -100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on ß') away from downstream DNA (on ß) to increase the Cy3-Cy5 distance by ∼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3-Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Quantifying Interactions of Nucleobase Atoms with Model Compounds for the Peptide Backbone and Glutamine and Asparagine Side Chains in Water.

Alkylureas display hydrocarbon and amide groups, the primary functional groups of proteins. To obtain the thermodynamic information that is needed to analyze interactions of amides and proteins with nucleobases and nucleic acids, we quantify preferential interactions of alkylureas with nucleobases differing in the amount and composition of water-accessible surface area (ASA) by solubility assays. Using an established additive ASA-based analysis, we interpret these thermodynamic results to determine interactions of each alkylurea with five types of nucleobase unified atoms (carbonyl sp2O, amino sp3N, ring sp2N, methyl sp3C, and ring sp2C). All alkylureas interact favorably with nucleobase sp2C and sp3C atoms; these interactions become more favorable with an increasing level of alkylation of urea. Interactions with nucleobase sp2O are most favorable for urea, less favorable for methylurea and ethylurea, and unfavorable for dialkylated ureas. Contributions to overall alkylurea-nucleobase interactions from interactions with each nucleobase atom type are proportional to the ASA of that atom type with proportionality constant (interaction strength) α, as observed previously for urea. Trends in α-values for interactions of alkylureas with nucleobase atom types parallel those for corresponding amide compound atom types, offset because nucleobase α-values are more favorable. Comparisons between ethylated and methylated ureas show interactions of amide compound sp3C with nucleobase sp2C, sp3C, sp2N, and sp3N atoms are favorable while amide sp3C-nucleobase sp2O interactions are unfavorable. Strongly favorable interactions of urea with nucleobase sp2O but weakly favorable interactions with nucleobase sp3N indicate that amide sp2N-nucleobase sp2O and nucleobase sp3N-amide sp2O hydrogen bonding (NH···O═C) interactions are favorable while amide sp2N-nucleobase sp3N interactions are unfavorable. These favorable amide-nucleobase hydrogen bonding interactions are prevalent in specific protein-nucleotide complexes.

The mechanism and high-free-energy transition state of lac repressor-lac operator interaction.

Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)-lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO.

Experimental Atom-by-Atom Dissection of Amide-Amide and Amide-Hydrocarbon Interactions in H2O.

Quantitative information about amide interactions in water is needed to understand their contributions to protein folding and amide effects on aqueous processes and to compare with computer simulations. Here we quantify interactions of urea, alkylated ureas, and other amides by osmometry and amide-aromatic hydrocarbon interactions by solubility. Analysis of these data yields strengths of interaction of ureas and naphthalene with amide sp2O, amide sp2N, aliphatic sp3C, and amide and aromatic sp2C unified atoms in water. Interactions of amide sp2O with urea and naphthalene are favorable, while amide sp2O-alkylurea interactions are unfavorable, becoming more unfavorable with increasing alkylation. Hence, amide sp2O-amide sp2N interactions (proposed n-σ* hydrogen bond) and amide sp2O-aromatic sp2C (proposed n-π*) interactions are favorable in water, while amide sp2O-sp3C interactions are unfavorable. Interactions of all ureas with sp3C and amide sp2N are favorable and increase in strength with increasing alkylation, indicating favorable sp3C-amide sp2N and sp3C-sp3C interactions. Naphthalene results show that aromatic sp2C-amide sp2N interactions in water are unfavorable while sp2C-sp3C interactions are favorable. These results allow interactions of amide and hydrocarbon moieties and effects of urea and alkylureas on aqueous processes to be predicted or interpreted in terms of structural information. We predict strengths of favorable urea-benzene and N-methylacetamide interactions from experimental information to compare with simulations and indicate how amounts of hydrocarbon and amide surfaces buried in protein folding and other biopolymer processes and transition states can be determined from analysis of urea and diethylurea effects on equilibrium and rate constants.

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase.

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ßß'ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

Basis of Protein Stabilization by K Glutamate: Unfavorable Interactions with Carbon, Oxygen Groups.

Potassium glutamate (KGlu) is the primary Escherichia coli cytoplasmic salt. After sudden osmotic upshift, cytoplasmic KGlu concentration increases, initially because of water efflux and subsequently by K+ transport and Glu- synthesis, allowing water uptake and resumption of growth at high osmolality. In vitro, KGlu ranks with Hofmeister salts KF and K2SO4 in driving protein folding and assembly. Replacement of KCl by KGlu stabilizes protein-nucleic acid complexes. To interpret and predict KGlu effects on protein processes, preferential interactions of KGlu with 15 model compounds displaying six protein functional groups-sp3 (aliphatic) C; sp2 (aromatic, amide, carboxylate) C; amide and anionic (carboxylate) O; and amide and cationic N-were determined by osmometry or solubility assays. Analysis of these data yields interaction potentials (α-values) quantifying non-Coulombic chemical interactions of KGlu with unit area of these six groups. Interactions of KGlu with the 15 model compounds predicted from these six α-values agree well with experimental data. KGlu interactions with all carbon groups and with anionic (carboxylate) and amide oxygen are unfavorable, while KGlu interactions with cationic and amide nitrogen are favorable. These α-values, together with surface area information, provide quantitative predictions of why KGlu is an effective E. coli cytoplasmic osmolyte (because of the dominant effect of unfavorable interactions of KGlu with anionic and amide oxygens and hydrocarbon groups on the water-accessible surface of cytoplasmic biopolymers) and why KGlu is a strong stabilizer of folded proteins (because of the dominant effect of unfavorable interactions of KGlu with hydrocarbon groups and amide oxygens exposed in unfolding).

Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9.

In vitro, replacing KCl with potassium glutamate (KGlu), the Escherichia coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu- in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to the effects of other stabilizers (KCl, KF, and K2SO4) and destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 ° C, performed as a function of the concentration of KGlu or another salt and monitored by NTL9 fluorescence, are analyzed to obtain R-values quantifying the Hofmeister salt concentration (m3) dependence of the unfolding equilibrium constant K(obs) [r-value = −d ln K(obs)/dm3 = (1/RT) dΔG(obs) ° /dm3 = m-value/RT]. r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 ° C, we determine a KGlu r-value of 3.22 m(−1) and K2SO4, KF, KCl, GuHCl, and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m(−1), respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 ° C.

Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA Polymerase-λP(R) Promoter Complexes.

Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.

Contributions of Coulombic and Hofmeister Effects to the Osmotic Activation of Escherichia coli Transporter ProP.

Osmosensing transporters mediate osmolyte accumulation to forestall cellular dehydration as the extracellular osmolality increases. ProP is a bacterial osmolyte-H(+) symporter, a major facilitator superfamily member, and a paradigm for osmosensing. ProP activity is a sigmoid function of the osmolality. It is determined by the osmolality, not the magnitude or direction of the osmotic shift, in cells and salt-loaded proteoliposomes. The activation threshold varies directly with the proportion of anionic phospholipid in cells and proteoliposomes. The osmosensory mechanism was probed by varying the salt composition and concentration outside and inside proteoliposomes. Data analysis was based on the hypothesis that the fraction of maximal transporter activity at a particular luminal salt concentration reflects the proportion of ProP molecules in an active conformation. ProP attained the same activity at the same osmolality when diverse, membrane-impermeant salts were added to the external medium. Contributions of Coulombic and/or Hofmeister salt effects to ProP activation were examined by varying the luminal salt cation (K(+) and Na(+)) and anion (chloride, phosphate, and sulfate) composition and then systematically increasing the luminal salt concentration by increasing the external osmolality. ProP activity increased with the sixth power of the univalent cation concentration, independent of the type of anion. This indicates that salt activation of ProP is a Coulombic, cation effect resulting from salt cation accumulation and not site-specific cation binding. Possible origins of this Coulombic effect include folding or assembly of anionic cytoplasmic ProP domains, an increase in local membrane surface charge density, and/or the juxtaposition of anionic protein and membrane surfaces during activation.

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure.

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >10(5)-fold and >10(2)-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ(70) region 1.1 (σ1.1), ß' jaw and ß' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases -4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either ß' jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.

Initial events in bacterial transcription initiation.

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.