Tryptophan Regulated Attenuation Protein (TRAP)

Caroline Burley '17 and Natalie Plick '16


I. Introduction

While absolutely essential, gene expression is a costly cellular process. As a means of conserving energy and cellular resources, living organisms have evolved mechanisms to regulate gene expression.

Often, protein products are involved in their own transcriptional regulation. In B. subtilis, tryptophan mediates transcription and translation of the genes responsible for its biosynthesis. Tryptophan accomplishes this through interactions with a regulatory protein known as tryptophan-activated RNA binding attenuation protein (TRAP). When bound by tryptophan, TRAP binds the leader sequence of trp-mRNA, inducing a conformational change in the mRNA. This change prevents further transcription of the tryptophan operon.

Alternately, when tryptophan is low in the cell, a second protein, anti-TRAP protein (AT) is activated. AT is primarily activated by uncharged trp tRNA, the abundance of which is another indication of low cellular tryptophan. AT binds TRAP, thereby preventing it from binding trp mRNA, and allowing the operon to be transcribed.  

Here we will focus on interactions between TRAP and tryptophan, RNA, and anti-TRAP.

II. General Structure

TRAP is a ring-shaped protein 85 angstroms in diameter and with a 23 angstrom hole in the middle. TRAP is composed of 11 identical subunits: A B C D E F G H I J K. Each subunit is comprised of 7 antiparallel beta sheets (A-G) . Extensive hydrogen bonds between adjacent subunits stabilize TRAP's quaternary structure .

When complexed with tryptophan and RNA, TRAP generally exists as a stacked tetramer TRAP Tetramer. The two inner TRAP proteins interact with tryptophan and RNA, whereas the two outer proteins only interact with TRAP and the solvent. Here we present half of the tetramer: an outer TRAP complexed with an inner TRAP .

III. L-Tryptophan Binding

TRAP is stabilized by interactions with tryptophan. One tryptophan molecule binds between each of the eleven TRAP subunits . When bound  to TRAP, the indole ring of tryptophan sits in a non-polar binding pocket, formed by residues from the two neighboring subunits. Additionally, the nitrogen on tryptophan's indole ring hydrogen bonds with a carbonyl oxygen of Gln 47. Tryptophan's amino and carbonyl termini bind to TRAP through eight hydrogen bonds. These bonds are formed with residues from two adjacent subunits (Thr 25, Gly 27, Ala 28, Thr 30, Thr 49, Thr 52, Ser 53, Gln 47) .

IV. RNA Binding

When activated by tryptophan binding, TRAP binds the leader sequence of RNA, which proceeds the structural genes of the trp operon. When tryptophan-bound TRAP binds RNA it causes a hairpin structure to form. This hairpin structure is termed the "terminator structure" because it causes RNA polymerase to release the RNA before the operon is fully transcribed.

TRAP's RNA target binds in a single stranded state, wrapping around TRAP's perimeter. Most interactions between RNA and TRAP occur between the RNA's bases rather than its phosphate backbone. Specifically, RNA binds TRAP through eleven tri-nucleotide (G/U1, A2, G3) repeats


G1 interacts with Lys 37's aliphatic chain through Van der Waal forces . G1's amine group hydrogen bonds with Asp 39's carbonyl oxygen .


A2 binds TRAP through three hydrogen bonds: one between its sugar hydroxyl and Thr 30's carbonyl oxygen (via a water molecule); another between adenine's exocyclic amino group and Lys 37's main chain carbonyl oxygen; and a third between adenine's N1 and Lys 37's main chain amine group   . Additionally, A2, G3, and Phe 32 are nearly parallel to one another and interact through ring stacking .


G3 further interacts with the protein through 6 hydrogen bonds, most of which are between guanine and TRAP. Through its carbonyl oxygen, the base hydrogen bonds to Lys 56 and the amine side chain of Arg 58 . Glu 36 accepts two hydrogen bonds from guanine, each coming from a nitrogen . Furthermore, guanine's amine group shares a single hydrogen bond with Thr 30's main chain carbonyl . Finally, guanine's ribose 2' hydroxyl hydrogen bonds with Phe 32's main chain amine group .

V. Anti-TRAP Binding

AT is expressed when levels of charged trp-tRNA are low, an indication of low cellular tryptophan. Under such conditions, AT binds TRAP, sterically preventing it from binding RNA. This allows the trp operon to be expressed .

AT trimers bind the TRAP protein. Each AT trimer covers exactly 2 TRAP subunits such that wild type TRAP can accommodate no more than 5 AT trimers. Of the three AT trimers, the majority of the TRAP-AT contacts form between a single AT chain and one TRAP subunit. While another chain of the trimer interacts less extensively with an adjacent TRAP subunit, and the final chain of the trimer interacts with the solvent .

While details regarding TRAP-AT interactions are still being elucidated, targetted mutagenesis has revealed several important residues. As one might expect, many of the residues necessary for TRAP-RNA interactions also play an integral role in TRAP-AT binding. TRAP's Phe 32--which normally has ring stacking interactions with  RNA bases--occupies AT's non-polar binding pocket . Additionally, TRAP's Lys 37--which often engages in Van der Waal forces and H-bonds with RNA--forms a salt bridge with Asp residues in AT . Finally, two Arg 58's of neighboring TRAP subunits form non-polar interactions with AT .

VI. References

Yanofsky, C. RNA-based Regulation of Genes of Tryptophan Synthesis and Degradation, in Bacteria. 2007. RNA, 13 (8): 1141-1154.

Antson, A. A., Otridge, J., Brzozowski, A.M., Dodson, E. J., Dodson, G.G., Wilson, K.S., Smith T.M., Yang, M., Kurecki, T., Gollnick, P. The Structure of the trp RNA-binding Attenuation Protein. 1995. Nature, 374: 693-700.

Antson, A. A., Dodson, E.J., Dodson, G., Graves, R.B., Chen, X., and Gollnick, P. 1999. The Structure of trp RNA-binding Attenuation Protein, TRAP, bound to RNA. Nature, 401: 235-241.

Hopcroft, N.H., Manfredo, A., Wendt, A.L., Brzozowski, A.M., Gollnick, P., Antson, A.A. 2004. The interaction of RNA with TRAP: The Role of Triplet Repeats and Separating Spacer Nucleotides. J. Mol. Bio., 338: 43-53.

Watanabe, M., Heddle J.G., Kikuchi, K., Unzai, S., Akashi, S., Park, S., and Tame, J.R.H. 2009. The Nature of the TRAP-Anti-TRAP Complex. Proc. Natl. Acad. Sci. USA., 106 (7): 2176-2181.

Shevtsov, M.B., Chen Y., Gollnick, P., Antson, A.A., 2005. Crystal Structure of Bacillus Subtilis Anti-TRAP Protein, an Antagonist of TRAP/RNA Interaction. Proc. Natl. Acad. Sci. USA, 102 (49): 17600-17605.

Antson, A. (1999). "Packing of Molecules in the Crystal." [Online Image] Structure of the trp RNA-Binding Attenuation Protein, TRAP, Bround to RNA. Nature. Retrieved from:

Excess trp. Digital image. Technische Universitat Muchen. Web. 1 Dec. 2014.

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