Structural Probing of the T Box Antiterminator-tRNA Complex

Abstract

Structural changes in a unique RNA-RNA binding interaction were probed using 2-aminopurine. The 5′-untranslated leader region (5′-UTR) of the T box family of genes folds into a structure that selectively recognizes a specific tRNA through two unique base-pairing events. The first involves base pairing between the anticodon of cognate tRNA and a tri-nucleotide sequence (specifier sequence) in the specifier loop of stem 1 in the 5′-UTR. The second base pairing event involves the non-aminoacylated tRNA acceptor end base pairing with the first four nucleotides at the 5′-end of a bulge in a highly conserved antiterminator element. In the absence of the stabilization of the uncharged tRNA acceptor end base pairing to the antiterminator element, the more thermodynamically stable terminator element forms and transcription terminates. In this manner, the leader region specifically recognizes cognate tRNA and responds structurally to the charging ratio of the tRNA to regulate transcription, thus making the T box mechanism an example of a riboswitch. Interestingly, the predicted thermodynamic stabilization provided by the four base pairs between the tRNA acceptor end and the antiterminator is not sufficient to overcome the predicted stability difference between the antiterminator and the terminator elements. Consequently, additional structural factors likely play a role in stabilizing the resulting complex. The structural changes induced in both the antiterminator element and the tRNA were investigated using a model system to determine what additional factors, beyond base pairing, contribute to stabilization of the resulting tRNA-antiterminator complex. Fluorescence monitoring of the base analog 2-aminopurine at select positions throughout a model complex indicated that binding results in an induced-fit and a highly stacked environment at the binding interface. These structural features contribute to the overall stabilization of the complex beyond the four base pairs.