Trp RNA-binding Attenuation Protein Research Articles

A Self‐Assembled Protein Nanotube with High Aspect Ratio

Production of a self-assembled protein nanotube achieved through engineering of the 11mer ring protein trp RNA-binding attenuation protein is described. The produced mutant protein is able to stack in solution to produce an extremely narrow, uniform nanotube apparently stabilized by a mixture of disulfide bonds and hydrophobic interactions. Assembly is reversible and the length of tube can potentially be controlled. Large quantities of hollow tubes 8.5 nm in overall diameter with lengths varying from 7 nm to over 1 microm are produced. The structure is analyzed using transmission electron microscopy, atomic force microscopy, mass spectrometry, and single-particle analysis and it is found that component rings stack in a head-to-head fashion. The internal diameter of the tube is 2.5 nm, and the amino acid residues lining the central cavity can be mutated, raising the possibility that the tube can be filled with a variety of conducting or semiconducting materials.

The nature of the TRAP–Anti-TRAP complex

Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-A resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.

RNA and Protein Complexes of trp RNA-Binding Attenuation Protein Characterized by Mass Spectrometry

We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution.

Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic ( trp) genes in response to changes in intracellular levels of free l-tryptophan in many Gram-positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT 3) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT 12) structure. Using alanine-scanning mutagenesis we found four residues, all located on the “top” region of AT 3, that are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation.

Insights into activation and RNA binding of trp RNA-binding attenuation protein (TRAP) through all-atom simulations

Tryptophan biosynthesis in Bacillus stearothermophilus is regulated by a trp RNA binding attenuation protein (TRAP). It is a ring-shaped 11-mer of identical 74 residue subunits. Tryptophan binding pockets are located between adjacent subunits, and tryptophan binding activates TRAP to bind RNA. Here, we report results from all-atom molecular dynamics simulations of the system, complementing existing extensive experimental studies. We focus on two questions. First, we look at the activation mechanism, of which relatively little is known experimentally. We find that the absence of tryptophan allows larger motions close to the tryptophan binding site, and we see indication of a conformational change in the BC loop. However, complete deactivation seems to occur on much longer time scales than the 40 ns studied here. Second, we study the TRAP-RNA interactions. We look at the relative flexibilities of the different bases in the complex and analyze the hydrogen bonds between the protein and RNA. We also study the role of Lys37, Lys56, and Arg58, which have been experimentally identified as essential for RNA binding. Hydrophobic stacking of Lys37 with the nearby RNA base is confirmed, but we do not see direct hydrogen bonding between RNA and the other two residues, in contrast to the crystal structure. Rather, these residues seem to stabilize the RNA-binding surface, and their positive charge may also play a role in RNA binding. Simulations also indicate that TRAP is able to attract RNA nonspecifically, and the interactions are quantified in more detail using binding energy calculations. The formation of the final binding complex is a very slow process: within the simulation time scale of 40 ns, only two guanine bases become bound (and no others), indicating that the binding initiates at these positions. In general, our results are in good agreement with experimental studies, and provide atomic-scale insights into the processes.

Using the Ring‐Shaped Protein TRAP to Capture and Confine Gold Nanodots on a Surface.

The cavity of the toroidal protein TRAP (trp RNA-binding attenuation protein) is modified to capture gold nanodots in solution. By engineering a titanium-binding peptide onto one surface of the ring it is also possible to bind it specifically and tightly to a TiO2 surface. TRAP bound in this way is then used to capture gold nanodots and attach them to prepared surfaces. Gold-protein complexes are observed using atomic force microscopy and transmission electron microscopy. The modified TRAP is used to build gold nanodots into the SiO2 layer of a metal oxide semiconductor. This is the first use of a ring protein, rather than the more commonly used spherical protein cages, to constrain nanodots to a surface. This method is an important addition to the current range of bionanotechnology tools and may be the basis for future, multicomponent electronic devices.

Dynamic Allostery in the Ring Protein TRAP

We have discovered distinct, characteristic differences in the thermodynamic signatures of tryptophan binding by trp RNA-binding attenuation protein (TRAP) from two different bacterial species. The TRAP 11mer ring binds 11 molecules of tryptophan at symmetry-related sites. Tryptophan binding to Bacillus stearothermophilus TRAP is not cooperative, but isothermal titration calorimetry shows that filling the first tryptophan binding sites of Bacillus subtilis TRAP has a marked effect on the thermodynamics of subsequent ligand binding. We have identified a single, conservative amino acid replacement (Ile to Leu) in B. subtilis TRAP that abolishes this effect, and suggest the initial ligand binding causes a change throughout the wild-type protein ring.

The Rate of TRAP Binding to RNA Is Crucial for Transcription Attenuation Control of the B. subtilis trp Operon

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic and transport genes in Bacillus subtilis in response to changes in the levels of intracellular tryptophan. Transcription of the trpEDCFBA operon is controlled by an attenuation mechanism involving two overlapping RNA secondary structures in the 5′ leader region of the trp transcript; TRAP binding promotes formation of a transcription terminator structure that halts transcription prior to the structural genes. TRAP consists of 11 identical subunits and is activated to bind RNA by binding up to 11 molecules of L-tryptophan. The TRAP binding site in the leader region of the trp operon mRNA consists of 11 (G/U)AG repeats. We examined the importance of the rate of TRAP binding to RNA for the transcription attenuation mechanism. We compared the properties of two types of TRAP 11-mers: homo-11-mers composed of 11 wild-type subunits, and hetero-11-mers with only one wild-type subunit and ten mutant subunits defective in binding either RNA or tryptophan. The hetero-11-mers bound RNA with only slightly diminished equilibrium binding affinity but with slower on-rates as compared to WT TRAP. The hetero-11-mers showed significantly decreased ability to induce transcription termination in the trp leader region when examined using an in vitro attenuation system. Together these results indicate that the rate of TRAP binding to RNA is a crucial factor in TRAP's ability to control attenuation.

The trp RNA‐binding attenuation protein (TRAP) of Bacillus subtilis regulates translation initiation of ycbK, a gene encoding a putative efflux protein, by blocking ribosome binding

Expression of the Bacillus subtilis tryptophan biosynthetic genes trpEDCFBA and trpG, as well as a putative tryptophan transport gene (trpP), are regulated in response to tryptophan by the trp RNA-binding attenuation protein (TRAP). TRAP regulates expression of these genes by transcription attenuation and translation control mechanisms. Here we show that TRAP also regulates translation of ycbK, a gene that encodes a protein with similarities to known efflux proteins. As a likely TRAP-binding site consisting of 11 NAG repeats overlaps the ycbK translation initiation region, experiments were carried out to determine whether TRAP regulates translation of ycbK. TRAP was observed to regulate expression of a ycbK'-'lacZ translational fusion 20-fold in response to tryptophan. Binding studies indicated that TRAP binds to the ycbK transcript with high affinity and specificity. Footprint studies revealed that the central seven triplet repeats were protected by bound TRAP, while toeprint results suggest that nine triplet repeats contribute to TRAP binding. Additional toeprint and in vitro translation analyses demonstrated that bound TRAP regulates YcbK synthesis by blocking ribosome binding. We also identified two dipeptide coding minigenes between the Shine-Dalgarno sequence and start codon of ycbK. Expression of one of the minigenes modestly interfered with translation of ycbK.

Thermodynamics of Tryptophan-Mediated Activation of the trp RNA-Binding Attenuation Protein

The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary-structural elements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced-fit conformational change are discussed.

Rounding up: Engineering 12-Membered Rings from the Cyclic 11-Mer TRAP

The protein TRAP (trp RNA binding attenuation protein) forms a highly thermostable ring-shaped 11-mer. By linking in tandem two, three, or four DNA sequences encoding TRAP monomers, we have engineered new rings that consist of 12 TRAP subunits and bind 12 ligand molecules. The hydrogen bonding pattern and buried surface area within and between subunits are essentially identical between the 11-mer and 12-mer crystal structures. Why do the artificial proteins choose to make single 12-mer rings? The 12-mer rings are highly sterically strained by their peptide linkers and far from thermostable. That proteins choose to adopt a strained conformation of few subunits rather than an unstrained one with 11 subunits demonstrates the importance of entropic factors in controlling protein-protein interactions in general.

1P044 Ligand-dissociation increases protein flexibility in trp RNA-binding attenuation protein (TRAP) : Molecular dynamics simulation study(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P044 Ligand-dissociation increases protein flexibility in trp RNA-binding attenuation protein (TRAP) : Molecular dynamics simulation study(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Strongly correlated structure of axial-symmetric proteins. III. Complexes with DNA/RNA

Three cases are considered of protein-DNA (or protein-RNA) complexes with a strongly correlated structure based on symmetry. In the first the symmetry of the nucleic acid is the determinant element, the second contains a dominant protein and an adaptive DNA/RNA and in the third a perturbed symmetry arises from elements of both components. The first situation is exemplified by the filamentous bacteriophage Pf1 in a low- and high-temperature state. The Pyrococcus abyssi Sm core and the trp RNA-binding attenuation protein are examples of the second situation. Finally, the nucleosome core particle represents the cooperative compromise between histone and DNA. In all the cases, the strong correlation in the structure is based on polygrammal scaling relations and on a molecular polygonal form lattice which depends on a single parameter.

Sensing complex regulatory networks by conformationally controlled hairpin ribozymes.

The hairpin ribozyme catalyses RNA cleavage by a mechanism utilizing its conformational flexibility during the docking of two independently folded internal loop domains A and B. Based on this mechanism, we designed hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides influencing the docking process. We incorporated a third domain C to assimilate alternate stable RNA motifs such as a pseudo-half-knot or an internal stem-loop structure. Small sequence changes in domain C allowed targeted switching of ribozyme activity: the same effector oligonucleotide can either serve as an inducer or repressor. The ribozymes were applied to trp leader mRNA, the RNA sequence tightly bound by l-tryptophan-activated trp-RNA-binding attenuation protein (TRAP). When domain C is complementary to this mRNA, ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. This approach allows to precisely sense the activity status of a protein controlled by its metabolite molecule.

Interaction of the trp RNA-binding Attenuation Protein (TRAP) with Anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5′-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA Trp induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP–AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of ∼3×10 14 M −3. About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP–AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.

The Interaction of RNA with TRAP: The Role of Triplet Repeats and Separating Spacer Nucleotides

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes in several Bacilli by binding to the leader region of the nascent trp mRNA, inhibiting continued transcription. The 11 subunit TRAP molecule is active in complex with tryptophan, and binds consequently an RNA target segment consisting of 11 (G/U)AG triplets, each separated by two or three non-conserved “spacer” nucleotides. Here, we report the first crystal structures of TRAP in a complex with RNA containing UAG triplets separated by two nucleotides and in a complex with RNA containing GAG triplets separated by three nucleotides. Comparison with known structures of TRAP–RNA complexes shows that both substitution of G-1 with U-1 in the triplet and addition of an extra spacer nucleotide lead to a more flexible complex. This suggests an explanation why, in the trp leader RNA, all three-nucleotide spacer regions are followed by a G-1 nucleotide. Taken together, the structures demonstrate that RNA binding to TRAP is mediated by specific interactions involving the A-2 and G-3 nucleotides of the triplet. This is accompanied by the disruption of stacking interactions between the bases of the other nucleotides, contributing to the increase in entropy that drives binding.

Characterization of a trp RNA-binding Attenuation Protein (TRAP) Mutant with Tryptophan Independent RNA Binding Activity

TRAP ( trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism ( trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.

A Mg2+-dependent RNA Tertiary Structure Forms in the Bacillus subtilis trp Operon Leader Transcript and Appears to Interfere with trpE Translation Control by Inhibiting TRAP Binding

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg2+-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5′-p-azidophenacyl-containing transcripts revealed a Mg2+-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg2+ inhibits TRAP binding, which may be partially due to the effect of Mg2+ on RNA tertiary structure formation. Results from expression studies using trpE′-′lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg2+-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA.

The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon. Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp). Here we show that translation of AT also is regulated by uncharged tRNATrp. A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene. Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production. Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression.

TRAP–RNA Interactions Involved in Regulating Transcription Attenuation of the Bacillus subtilis trp Operon

This chapter describes the methods that have been used to characterize the interaction of trp RNA-binding attenuation protein (TRAP) with RNA, and to study how these interactions are involved in regulating transcription attenuation of the trp operon. TRAP is encoded by mtrB, the second gene of the mtrAB operon. MtrA encodes GTP cyclohydrolase I, an enzyme involved in folic acid biosynthesis. Both Bacillus subtilis and Bacillus stearothermophilus TRAP have been expressed in Escherichia coli (E.Coli) using the T7 expression system. Several assays have been used to examine the interaction of TRAP with RNA. These assays use 32 P-labeled transcripts containing either the trp leader sequence or artificial TRAP binding sites. One of the quickest and most reliable methods to quantitatively analyze TRAP binding to RNA is the nitrocellulose filter binding assay. Equilibrium dialysis was first used to demonstrate that tryptophan binds to TRAP using an assay. This simple method involves placing TRAP on one side of a dialysis membrane and [ 14 C] L-tryptophan on the other side and then allowing dialysis to proceed until equilibrium is reached. Both transcriptional and translational gene fusions containing the trp promoter and leader region fused to lacZ have been used extensively to examine TRAP function in vivo.