T box transcription antitermination riboswitch: influence of nucleotide sequence and orientation on tRNA binding by the antiterminator element

Abstract

Many bacteria utilize riboswitch transcription regulation to monitor and appropriately respond to cellular levels of important metabolites or effector molecules. The T box transcription antitermination riboswitch responds to cognate uncharged tRNA by specifically stabilizing an antiterminator element in the 5'-untranslated mRNA leader region and precluding formation of a thermodynamically more stable terminator element. Stabilization occurs when the tRNA acceptor end base pairs with the first four nucleotides in the seven nucleotide bulge of the highly conserved antiterminator element. The significance of the conservation of the antiterminator bulge nucleotides that do not base pair with the tRNA is unknown, but they are required for optimal function. In vitro selection was used to determine if the isolated antiterminator bulge context alone dictates the mode in which the tRNA acceptor end binds the bulge nucleotides. No sequence conservation beyond complementarity was observed and the location was not constrained to the first four bases of the bulge. The results indicate that formation of a structure that recognizes the tRNA acceptor end in isolation is not the determinant driving force for the high phylogenetic sequence conservation observed within the antiterminator bulge. Additional factors or T box leader features more likely influenced the phylogenetic sequence conservation.

Figure 1

T box riboswitch and model RNAs. a) Schematic of T box untranslated leader mRNA recognition of cognate uncharged tRNA [4, 5]. The tRNA binds the leader region via base pairing of the anticodon with the Specifier Sequence and base pairing of the uncharged tRNA acceptor end nucleotides with the first 4 nucleotides in the antiterminator bulge. In the absence of tRNA binding, as transcription continues, a more stable stem-loop terminator element is formed and transcription is terminated (inset). The 5′ side of the terminator stem is comprised of nucleotides at the 3′ end of the antiterminator making the two elements mutually exclusive (bold line). b) T box antiterminator model RNA AM1A (positions that base pair with tRNA shown in grey); positions 2-15 of AM1A correspond to the highly conserved 14 nucleotide T box sequence, c) tRNA-UCCA, d) 3-AP-mh-UCCA model tRNA (discriminator base change corresponding to the single mismatch model 3-AP-mh-ACCA indicated by an arrow), e) randomized antiterminator RNA used for selection.

Figure 2

In vitro selection and selected sequences a) In vitro selection strategy, b) Cloned in vitro selected sequences from cycle 8 shown 5′-3′. Randomized (or original for AM1A) bulge nucleotide positions shown in black, non-randomized helical and 5′-extension (see text) regions shown in gray. Consensus sequence alignments are highlighted in green with number of duplicate sequences indicated in parentheses.

Figure 3

Selected sequences assayed for model tRNA binding. Nucleotides with anti-parallel complementarity to tRNA acceptor end shown in grey. Sequences correspond to those in Figure 2, but with the nine-nucleotide primer site sequence used for the in vitro selection deleted (designated by “Δ”, see text).