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From Proteopedia

The Tryptophan (Trp) RNA-binding attenuation protein (TRAP) is encoded by the mtrB gene of Bacillus subtilis, which is the second gene of the mtrAB operon^[1]. This protein is composed of eleven identical subunits which form a beta wheel and are linked by beta sheets^[2]. Each subunit can bind to a tryptophan molecule which induces conformational changes that allow the specific recognition and interaction with RNA. This mechanism is part of the regulation tryptophan synthesis in Bacillus Subtilis.

Functions

Regulation of the Trp operon

TRAP senses the intracellular concentration of tryptophan and then regulates the expression of the trpEDCFBA operon^[1].This operon, known as the tryptophan operon, contains a cluster of genes (Trp E, Trp D, Trp C, Trp F, Trp B, Trp A) that leads to the synthesis of the tryptophan synthetase, an enzyme that uses chorismic acid to produce tryptophan. All these genes are under the control of the same promoter. Hence TRAP is considered as an attenuation protein at the transcriptional level^[2]. When the intracellular level of tryptophan is high enough, TRAP binds to the leader sequence at the 5' end of the nascent RNA and induces the formation of a terminator hairpin which results in the termination of the operon's transcription^[1]. However, when the concentration of tryptophan is low TRAP can not interact with the RNA and the formation of an anti-terminator structure occurs which prevents the transcription from stopping^[1].

Regulation of the TrpE gene

TRAP also regulates the translation of the tryptophan operon. The protein is able to repress the translation of TrpE which is the first gene of this operon and which normally leads to the synthesis of the first protein of the tryptophan biosynthesis pathway, the anthranilate synthetase. The repression occurs when TRAP directly binds to the ribosome binding site and thus inhibits the initiation of the translation^[1]. The ribosome and TRAP are binding competitively to the RBS. When TRAP binds to the ARN of the trp operon, a RNA conformational change takes place and the trpE S-D sequence forms a hairpin, leading to the inhibition of the translation. The formation of this hairpin is also thought to be regulating trpD via translational coupling^[2].

Other functions

Not only TRAP has a role at both transcriptional and translational level of regulation of the Trp operon expression, but it has also a role on some other factors such as pabA, a protein involved in the synthesis of p-aminobenzoate, trpP, a tryptophan transport gene and ycbK, a gene encoding for an uncharacterized transporter. It regulates the translation of these genes by blocking the RBS^[2]. TRAP binds to the RNA segments which have the same functions as the S-D sequence in the trpE or also binds to the translation initiation domains^[2].

Mechanisms

The protein is formed by eleven identical chains, forming a doughnut-like structure called β-wheel. This structure contains an eleven fold symmetry axis between chains facing to each others, the total ring has a diameter of 23 Å and is 36 Å thick^[2]. is 75 amino-acids long and consists of a three-stranded and a four stranded antiparallel β-sheet. The three-stranded sheet of a subunit interacts with the four-stranded one of the adjacent chain to form a seven-stranded sheet ^[3]. This structure can be compared to structures found in immunoglobulins even if the connections between the beta-strands aren’t related^[2].

Tryptophan binding

The protein can bind eleven L-tryptophan molecule, each one binds to an formed between two adjacent subunits and it is covered by the . The subunits are linked by hydrophobic interactions and the tryptophan binding reinforces the quaternary structure by adding new hydrophobic interactions and hydrogen bonds between the adjacent subunits^[2]. The amino and carboxy functions of tryptophane form 8 hydrogen bonds bonds with the protein and its carboxyl oxygen interacts with one water molecule. This is the only water molecule in the hydrophobic pocket, indeed tryptophane is totally isolated from solvent thanks to the conformation change induced its binding that allows the closing of the pocket^[2]. The binding is highly cooperative, indeed is in contact with two consecutive tryptophan molecules, thus the binding of a first molecule modifies the loop conformation and promotes the fixation of a second one^[1].

Interaction with RNA

When tryptophan binds to a subunit of the protein it induces a conformational change between residues 25-33 and 49-52. This conformational change allows the binding of the target messenger RNA in . The interaction is specific, indeed the protein recognizes two characteristic regions of ten nucleotides called DR1 and DR2 between positions +36 and +91 of the leader transcript^[2]. The recognition requires multiple (G/U)AG patterns spaced by two or three nucleotides^[2]. The adenine and the guanine at the third position of this pattern are packed against the Phe32 sidechain. These bases are also involved in an intricated network of interactions with the side chains of the aminoacids of TRAP: the adenosine forms a hydrogen bond with and the guanine with ^[4]. Only the first nucleotide (U or G) forms a single hydrogen bond with the sidechain of an aspartate residue^[3]. The backbone of the RNA points out of the circle, and even if it only interacts with the protein by a single hydrogen bond formed by the 2’-OH of the guanosine at the third position of each pattern, it allows the specific recognition of RNA^[1]. However, if this interaction is conserved, it is possible to replace the others ribonucleotides by deoxyribonucleotides without altering the affinity of the binding ^[5]. These characteristics of RNA recognition and binding car be compared with the protein U1A of the spliceosome in interaction with snRNA ^[6]. Footprint assays with different fragments of the leader region have shown that RNA binding to TRAP is oriented, indeed the bases triplets at the 5’ end of the RNA bind first and are then followed by those at the 3’ end. It is yet unclear why ^[5].

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5 1.6} Babitzke P. Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein. Curr Opin Microbiol. 2004 Apr;7(2):132-9. PMID:15063849 doi:http://dx.doi.org/10.1016/j.mib.2004.02.003
2. ↑ ^{2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10} Antson AA, Otridge J, Brzozowski AM, Dodson EJ, Dodson GG, Wilson KS, Smith TM, Yang M, Kurecki T, Gollnick P. The structure of trp RNA-binding attenuation protein. Nature. 1995 Apr 20;374(6524):693-700. PMID:7715723 doi:http://dx.doi.org/10.1038/374693a0
3. ↑ ^{3.0 3.1} Muto Y, Oubridge C, Nagai K. RNA-binding proteins: TRAPping RNA bases. Curr Biol. 2000 Jan 13;10(1):R19-21. PMID:10660288
4. ↑ Antson AA, Dodson EJ, Dodson G, Greaves RB, Chen X, Gollnick P. Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA. Nature. 1999 Sep 16;401(6750):235-42. PMID:10499579 doi:10.1038/45730
5. ↑ ^{5.0 5.1} Barbolina MV, Li X, Gollnick P. Bacillus subtilis TRAP binds to its RNA target by a 5' to 3' directional mechanism. J Mol Biol. 2005 Jan 28;345(4):667-79. PMID:15588817 doi:http://dx.doi.org/10.1016/j.jmb.2004.10.071
6. ↑ Oubridge C, Ito N, Evans PR, Teo CH, Nagai K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature. 1994 Dec 1;372(6505):432-8. PMID:7984237 doi:http://dx.doi.org/10.1038/372432a0