The structure of trp RNA-binding attenuation protein

Antson, A.A.; Otridge, John; Brzozowski, A.M.; Dodson, E.J.; Dodson, Guy; Wilson, K.S.; Smith, Thomas M.; Yang, Min; Kurecki, Tomasz; Gollnick, Paul

doi:10.1038/374693a0

Cited by 200 publications

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“…The crystal structures of tryptophan-activated Bacillus stearothermophilus TRAP in binary complex with tryptophan (1QAW) (12) and in ternary complex with tryptophan and RNA (1C9S) (13,14) reveal an oligomer of eleven identical subunits related by an eleven-fold rotational axis of symmetry. The structure is comprised of eleven anti-parallel beta-sheets, with each sheet consisting of three strands from one subunit and four strands from the neighboring subunit ( Figure 1).…”

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“…The structure is comprised of eleven anti-parallel beta-sheets, with each sheet consisting of three strands from one subunit and four strands from the neighboring subunit ( Figure 1). The tryptophan ligand is completely buried between the beta-sheets at the interface between subunits in a cavity that is largely apolar with a few polar side-chain and backbone moieties making hydrogen bond contacts to the sidechain amide and backbone amino and carboxyl groups of the tryptophan (12,(14)(15)(16). The bound RNA wraps around the protein with conserved bases making specific contacts with TRAP residues (13,(16)(17)(18)(19).…”

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

et al. 2006

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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. Keywordscalorimetry; binding-coupled protein folding; allosteric regulation; oligomer; trp RNA-binding attenuation protein; induced fit; pre-existing conformational equilibriumThe undecameric (11-mer) trp RNA-binding attenuation protein (TRAP) is responsible for controlling the transcription (1-5), and in some cases the translation (6-11), of the genes responsible for tryptophan biosynthesis in many Bacilli. Transcriptional regulation of the trp operon in these Bacilli is achieved through attenuation, in which competing secondarystructural elements in the 5′ leader region of the nascent mRNA control the extent of transcriptional read-through of the structural genes (3-5). TRAP exercises transcriptional control by influencing the formation of these secondary-structural elements through tryptophan-dependent binding to the RNA. When tryptophan is limiting, TRAP is inactive and * Corresponding author contact information: Phone: 614-292-1377, FAX: 614-292-6773, Email: foster.281@osu does not bind to the RNA. This allows a stable anti-terminator hairpin to form, promoting transcriptional read-through of the entire operon. However, when the intracellular tryptophan level is sufficiently high, TRAP binds to tryptophan and becomes activated to bind to eleven triplet repeats of (G/U)AG's present in the 5′ leader region of the mRNA. When TRAP is bound, the anti-terminator RNA structure cannot form, allowing preferential formation of the term...

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

et al. 2006

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“…The trp RNA-binding Attenuation Protein (TRAP) is an RNA-binding protein that negatively regulates expression of the tryptophan biosynthesis (trp) genes in Bacillus subtilis in response to intracellular levels of tryptophan (Gollnick, 1994;Babitzke, 1997)+ Six of the seven trp genes are clustered in the trpEDCFBA operon+ Transcription of this operon is controlled by an attenuation mechanism involving two mutually exclusive RNA secondary structures that form in the leader region 59 to the structural genes+ One of these structures is an intrinsic transcription terminator and the other overlapping structure is an antiterminator+ When activated by binding tryptophan, TRAP binds to a series of 11 triplet repeats composed of GAG or UAG that, in part, overlap a portion of the antiterminator+ TRAP binding therefore prevents antiterminator formation, which leads to formation of the terminator, and transcription halts in the leader region+ Under conditions of limiting tryptophan, TRAP does not bind RNA and the antiterminator structure is favored, allowing transcription of the operon+ TRAP also regulates translation of several genes+ By binding to the same site in the leader region of trp read-through mRNAs, TRAP induces formation of an RNA structure that sequesters the trpE ribosome binding site, thereby inhibiting translation initiation (Kuroda et al+, 1988;Merino et al+, 1995;Du & Babitzke, 1998)+ Translation of trpG, which is in the folate operon (Slock et al+, 1990;Yang et al+, 1995), and of yhaG, which is believed to encode a tryptophan transport protein (Sarsero et al+, 2000a), is also regulated by TRAP+ In these cases the TRAP binding site overlaps the ribosome binding site, and for trpG, Du et al+ (1998) demonstrated that TRAP competes directly with ribosomes for binding to this mRNA+…”

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The mechanism of RNA binding to TRAP: Initiation and cooperative interactions

Elliott

Gottlieb²,

Gollnick³

2001

RNA

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The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 29-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.

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“…TRAP also regulates translation of trpP, a gene that encodes an apparent tryptophan transporter (22,29), and ycbK, a gene that shares sequence homology to known efflux protein genes (23,28). TRAP consists of 11 identical subunits arranged in a single ring (2). When activated by tryptophan, 11 KKR motifs on the perimeter of TRAP interact with multiple NAG trinucleotide repeats in target transcripts, with a preference for N of G Ϸ U Ͼ A Ͼ C, thereby wrapping the RNA around TRAP's perimeter (1,6,32).…”

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Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

Yakhnin

Babitzke

2007

J Bacteriol

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Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG ShineDalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG -lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P pabB ). A new trpG promoter (P trpG ) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P pabB . We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P trpG and that transcripts originating from P trpG are not subject to translationmediated displacement of bound TRAP.

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The structure of trp RNA-binding attenuation protein

Cited by 200 publications

References 33 publications

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

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

The mechanism of RNA binding to TRAP: Initiation and cooperative interactions

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

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