Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis.

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM.

Homotropic cooperativity is widespread in biological regulation. The homo-oligomeric ring-shaped trp RNA binding attenuation protein (TRAP) from bacillus binds multiple tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the trp operon mRNA. Ligand-activated binding to this specific RNA sequence regulates downstream biosynthesis of Trp in a feedback loop. Characterized TRAP variants form 11- or 12-mer rings and bind Trp at the interface between adjacent subunits. Various studies have shown that a pair of loops that gate each Trp binding site is flexible in the absence of the ligand and become ordered upon ligand binding. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity we engineered dodecameric (12-mer) TRAP variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated one of the protomers such that only every other site was competent for Trp binding. Thermodynamic and structural studies using native mass spectrometry, NMR spectroscopy, and cryo-EM provided unprecedented detail into the thermodynamic and structural basis for the observed ligand binding cooperativity. Such insights can be useful for understanding allosteric control networks and for the development of new ones with defined ligand sensitivity and regulatory control.

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis.

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring a three-helix bundle, or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form has been shown to bind and inhibit TRAP. We demonstrate the utility of native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) for monitoring the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we report the use of solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP inhibition.

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand-dependent population distributions.

Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand-binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric trp RNA-binding attenuation protein (TRAP). First, we developed a nearest-neighbor statistical thermodynamic binding model comprising microscopic free energies for ligand binding to isolated sites ΔG0 , and for coupling between adjacent sites, ΔGα . Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored ligand-dependent population shifts using conventional spectroscopic and calorimetric methods and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity.

Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP.

Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding, it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding sites, because the observable [e.g., nuclear magnetic resonance (NMR) chemical shift changes, fluorescence, and enthalpy] can be altered by allostery, thereby distorting its proportionality to site occupancy. Native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. MS-compatible solutions did not significantly perturb protein structure or thermodynamics as assessed by isothermal titration calorimetry and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by nMS. The population distributions could not be explained by a noncooperative binding model but were described well by a mechanistic nearest-neighbor cooperative model. Nonlinear least-squares fitting yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites. This approach may be applied to quantify thermodynamic cooperativity in other ring-shaped proteins.

Serine 51 residue of Citrobacter freundii tyrosine phenol-lyase assists in C-α-proton abstraction and transfer in the reaction with substrate.

In the spatial structure of tyrosine phenol-lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l-tyrosine and 3-fluoro-l-tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4'-position of the leaving phenol group in the reaction with the natural substrate.

Cleavage of 3'-terminal adenosine by archaeal ATP-dependent RNA ligase.

Methanothermobacter thermoautotrophicus RNA ligase (MthRnl) catalyzes formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl termini of single-stranded RNAs. It can also react with RNA with a 3'-phosphate end to generate a 2',3'-cyclic phosphate. Here, we show that MthRnl can additionally remove adenosine from the 3'-terminus of the RNA to produce 3'-deadenylated RNA, RNA(3'-rA). This 3'-deadenylation activity is metal-dependent and requires a 2'-hydroxyl at both the terminal adenosine and the penultimate nucleoside. Residues that contact the ATP/AMP in the MthRnl crystal structures are essential for the 3'-deadenylation activity, suggesting that 3'-adenosine may occupy the ATP-binding pocket. The 3'-end of cleaved RNA(3'-rA) consists of 2',3'-cyclic phosphate which protects RNA(3'-rA) from ligation and further deadenylation. These findings suggest that ATP-dependent RNA ligase may act on a specific set of 3'-adenylated RNAs to regulate their processing and downstream biological events.

Mechanistic Models Fit to Variable Temperature Calorimetric Data Provide Insights into Cooperativity.

Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding it is necessary to define at a microscopic level the structural and thermodynamic consequences of binding of each ligand to its allosterically coupled site(s). However, dynamic sampling of alternative conformations (microstates) in allosteric molecules complicates interpretation of both structural and thermodynamic data. Isothermal titration calorimetry has the potential to directly quantify the thermodynamics of allosteric interactions, but usually falls short of enabling mechanistic insight. This is because 1) its measurements reflect the sum of overlapping caloric processes involving binding-linked population shifts within and between microstates, and 2) data are generally fit with phenomenological binding polynomials that are underdetermined. Nevertheless, temperature-dependent binding data have the potential to resolve overlapping thermodynamic processes, while mechanistically constrained models enable hypothesis testing and identification of informative parameters. We globally fit temperature-dependent isothermal titration calorimetry data for binding of 11 tryptophan ligands to the homo-undecameric trp RNA-binding Attenuation Protein from Bacillus stearothermophilus using nearest-neighbor statistical thermodynamic models. This approach allowed us to distinguish alternative nearest-neighbor interaction models, and quantifies the thermodynamic contribution of neighboring ligands to individual binding sites. We also perform conventional Hill equation modeling and illustrate how comparatively limited it is in quantitative or mechanistic value. This work illustrates the potential of mechanistically constrained global fitting of binding data to yield the microscopic thermodynamic parameters essential for deciphering mechanisms of cooperativity in a wide range of ligand-regulated homo-oligomeric assemblies.

Identification of a Residue (Glu60) in TRAP Required for Inducing Efficient Transcription Termination at the trp Attenuator Independent of Binding Tryptophan and RNA.

Transcription of the tryptophan (trp) operon in Bacillus subtilis is regulated by an attenuation mechanism. Attenuation is controlled by the trpRNA-binding attenuation protein (TRAP). TRAP binds to a site in the 5' leader region of the nascent trp transcript in response to the presence of excess intracellular tryptophan. This binding induces transcription termination upstream of the structural genes of the operon. In prior attenuation models, the role of TRAP was only to alter the secondary structure of the leader region RNA so as to promote formation of the trp attenuator, which was presumed to function as an intrinsic terminator. However, formation of the attenuator alone has been shown to be insufficient to induce efficient termination, indicating that TRAP plays an additional role in this process. To further examine the function of TRAP, we performed a genetic selection for mutant TRAPs that bind tryptophan and RNA but show diminished termination at the trp attenuator. Five such TRAP mutants were obtained. Four of these have substitutions at Glu60, three of which are Lys (E60K) substitutions and the fourth of which is a Val (E60V) substitution. The fifth mutant obtained contains a substitution at Ile63, which is on the same ß-strand of TRAP as Glu60. Purified E60K TRAP binds tryptophan and RNA with properties similar to those of the wild type but is defective at inducing termination at the trp attenuator in vitroIMPORTANCE Prior models for attenuation control of the B. subtilis trp operon suggested that the only role for TRAP is to bind to the leader region RNA and alter its folding to induce formation of an intrinsic terminator. However, several recent studies suggested that TRAP plays an additional role in the termination mechanism. We hypothesized that this function could involve residues in TRAP other than those required to bind tryptophan and RNA. Here we obtained TRAP mutants with alterations at Glu60 that are deficient at inducing termination in the leader region while maintaining tryptophan and RNA binding properties similar to those of the WT protein. These studies provide additional evidence that TRAP-mediated transcription termination at the trp attenuator is neither intrinsic nor Rho dependent.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5' to 3' Translocase in Transcription Termination of Vaccinia Early Genes.

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis.

An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl-AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5'-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl-AMP complex.

Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant.

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.

Solution NMR Experiment for Measurement of (15)N-(1)H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes.

NMR residual dipolar couplings (RDCs) are exquisite probes of protein structure and dynamics. A new solution NMR experiment named 2D SE2 J-TROSY is presented to measure N-H RDCs for proteins and supramolecular complexes in excess of 200 kDa. This enables validation and refinement of their X-ray crystal and solution NMR structures and the characterization of structural and dynamic changes occurring upon complex formation. Accurate N-H RDCs were measured at 750 MHz (1)H resonance frequency for 11-mer 93 kDa (2)H,(15)N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τc of 120 ns. This is about twice as long as that for the most slowly tumbling system, for which N-H RDCs could be measured, so far, and corresponds to molecular weights of ∼200 kDa at 25 °C. Furthermore, due to the robustness of SE2 J-TROSY with respect to residual (1)H density from exchangeable protons, increased sensitivity at (1)H resonance frequencies around 1 GHz promises to enable N-H RDC measurement for even larger systems.

The role of vaccinia termination factor and cis-acting elements in vaccinia virus early gene transcription termination.

Vaccinia virus early gene transcription termination requires the virion form of the viral RNA polymerase (vRNAP), Nucleoside Triphosphate Phosphohydrolase I (NPHI), ATP, the vaccinia termination factor (VTF), and a U5NU termination signal in the nascent transcript. VTF, also the viral mRNA capping enzyme, binds U5NU, and NPHI hydrolyzes ATP to release the transcript. NPHI can release transcripts independent of VTF and U5NU if vRNAP is not actively elongating. However, VTF and U5NU are required for transcript release from an elongating vRNAP, suggesting that the function of VTF and U5NU may be to stall the polymerase. Here we demonstrate that VTF inhibits transcription elongation by enhancing vRNAP pausing. Hence VTF provides the connection between the termination signal in the RNA transcript and viral RNA polymerase to initiate transcription termination. We also provide evidence that a second cis-acting element downstream of U5NU influences the location and efficiency of early gene transcription termination.

APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.

Characterization of TRAP-mediated regulation of the B. subtilis trp operon using in vitro transcription and transcriptional reporter fusions in vivo.

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism involving two alternative RNA secondary structures in the 5' leader region upstream of the structural genes. Regulation is accomplished, at least in part, by controlling which RNA structure forms during transcription of the operon. When intracellular tryptophan levels are high, the trp RNA-binding attenuation protein (TRAP) binds to the nascent trp mRNA to promote formation of a transcription terminator structure so as to induce transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind, the alternative antiterminator RNA structure forms, and the operon is transcribed. Several in vitro and in vivo assays have been utilized to study TRAP-mediated regulation of both transcription and translation. Here, we describe using in vitro transcription attenuation assays and in vivo trp-lacZ fusions to examine TRAP-mediated regulation of the trp genes.

Modulating TRAP-mediated transcription termination by AT during transcription of the leader region of the Bacillus subtilis trp operon.

An 11-subunit protein called trp RNA binding Attenuation Protein (TRAP) controls attenuation of the tryptophan biosynthetic (trpEDCFBA) operon in Bacillus subtilis. Tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the 5' leader region of trp mRNAs, and downregulates expression of the operon by promoting transcription termination prior to the structural genes. Anti-TRAP (AT) is an antagonist that binds to tryptophan-activated TRAP and prevents TRAP from binding to RNA, thereby upregulating expression of the trp genes. AT forms trimers, and multiple trimers bind to a TRAP 11mer. It is not known how many trimers must bind to TRAP in order to interfere with RNA binding. Studies of isolated TRAP and AT showed that AT can prevent TRAP from binding to the trp leader RNA but cannot dissociate a pre-formed TRAP-RNA complex. Here, we show that AT can prevent TRAP-mediated termination of transcription by inducing dissociation of TRAP from the nascent RNA when it has bound to fewer than all 11 (G/U)AG repeats. The 5'-most region of the TRAP binding site in the nascent transcript is most susceptible to dissociation from TRAP. We also show that one AT trimer bound to TRAP 11mer reduces the affinity of TRAP for RNA and eliminates TRAP-mediated transcription termination in vitro.

Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP.

The control of tryptophan production in Bacillus is a paradigmatic example of gene regulation involving the interplay of multiple protein and nucleic acid components. Central to this combinatorial mechanism are the homo-oligomeric proteins TRAP (trp RNA-binding attenuation protein) and anti-TRAP (AT). TRAP forms undecameric rings, and AT assembles into triskelion-shaped trimers. Upon activation by tryptophan, the outer circumference of the TRAP ring binds specifically to a series of tandem sequences present in the 5' UTR of RNA transcripts encoding several tryptophan metabolism genes, leading to their silencing. AT, whose expression is up-regulated upon tryptophan depletion to concentrations not exceeding a ratio of one AT trimer per TRAP 11-mer, restores tryptophan production by binding activated TRAP and preventing RNA binding. How the smaller AT inhibitor prevents RNA binding at such low stoichiometries has remained a puzzle, in part because of the large RNA-binding surface on the tryptophan-activated TRAP ring and its high affinity for RNA. Using X-ray scattering, hydrodynamic, and mass spectrometric data, we show that the polydentate action of AT trimers can condense multiple intact TRAP rings into large heterocomplexes, effectively reducing the available contiguous RNA-binding surfaces. This finding reveals an unprecedented mechanism for substoichiometric inhibition of a gene-regulatory protein, which may be a widespread but underappreciated regulatory mechanism in pathways that involve homo-oligomeric or polyvalent components.

The Bacillus subtilis TRAP protein can induce transcription termination in the leader region of the tryptophan biosynthetic (trp) operon independent of the trp attenuator RNA.

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. When intracellular tryptophan levels are high, the TRAP protein binds to the 5' leader region of the nascent trp mRNA and induces transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator) can form in the leader region RNA. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. However, recent studies have shown that the attenuator is not an effective intrinsic terminator. From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Hence we have further examined the role of the attenuator RNA in TRAP-mediated transcription termination. TRAP was found to cause efficient transcription termination in the trp leader region in vivo when the attenuator was mutated or deleted. However, TRAP failed to induce transcription termination at these mutant attenuators in a minimal in vitro transcription system with B. subtilis RNA polymerase. Further studies using this system showed that NusA as well as the timing of TRAP binding to RNA play a role in the observed differences in vivo and in vitro.