Fluorescence characterization of the transfer RNA-like domain of transfer messenger RNA in complex with small binding protein B.

Defective or truncated messenger RNAs (mRNAs) lacking stop codons lead to ribosome stalling on the end of the message. Stalled ribosomes can cause two major problems for the cell: production of incomplete, non-functional and potentially toxic proteins, and depletion of the ribosomal pool available for translation.1,2 In bacteria, these problems can be solved by a rescue process called trans-translation, which employs the hybrid transfer-messenger RNA (tmRNA) and its highly conserved cofactor, small protein B (SmpB).3,4 These two factors are universally conserved in bacterial species and trans-translation is important for a wide variety of physiological processes, including gene expression, pathogenesis, and response to stress, making tmRNA an attractive target for antibacterial drug discovery.1,5 Indeed, Pyrazinamide, a first-line tuberculosis drug, acts by inhibiting the ribosome rescue pathway.6 Transfer-messenger RNA consists of two domains (Figure 1A): the tRNA-like domain (TLD) and the mRNA-like domain (MLD). The TLD includes an acceptor stem and a TΨC arm (Figure 1A,B). However, in lieu of a D arm, it contains an irregular D loop without predicted helical pairing, and instead of an anticodon loop it contains an irregular helix (H2a) that joins the TLD to the MLD through four pseudoknots (pk1–pk4, Figure 1AB). The MLD comprises an open reading frame that encodes for a protein tag that signals for degradation of the incomplete protein.3,7–9 Transfer-messenger RNA enters into the A site of stalled ribosomes with its TLD in complex with SmpB and the elongation factor EF-Tu.3 In the A site, the nascent peptide chain is transferred to the TLD, which translocates into the P site releasing the defective mRNA. Then, the first codon of the MLD enters the decoding center, where it is translated by normal aminoacyl-tRNAs. The resulting peptide tag is recognized by specific cellular proteases that degrade the defective protein.1,8,10 All of these steps require a special set of interactions between tmRNA, SmpB, the ribosome and the translational cofactors.11 Figure 1 Secondary structure of tmRNA and the fluorophore labeled constructs A transient electric birefringence (TEB) study of the tmRNA TLD in absence of SmpB has revealed a more obtuse angle (110° instead of 90°) between the acceptor and helix H2a stems compared to canonical tRNAs.12 This has been confirmed in the crystal structure of the TLD-SmpB complex, which revealed an obtuse angle of 120° between the acceptor stem and the helix H2a, but a 90° angle between the acceptor stem and SmpB.13 Based on this result, the authors hypothesized that SmpB mimics a canonical acceptor stem and interacts with the decoding site, while the H2a arm functions as a class II tRNA long variable arm. More recently, interactions between SmpB and the decoding site have supported this hypothesis.14,15 It has been previously hypothesized that the lack of a D-stem in the TLD may provide flexibility in the elbow region of tmRNA, between H2 and the acceptor arm, which can be stabilized by its interaction with SmpB.16 This is an important question because it affects the possible tmRNA conformations during tmRNA accommodation and how the MLD is introduced into the decoding center while the aminoacyl group resides in the peptidyl transferase center. In particular, it is not yet known whether SmpB or Mg2+ ions modulate the TLD domain structure. To address this question, we have used fluorescence anisotropy and FRET to measure the distance between the acceptor and the H2a stem in the presence and absence of SmpB. We also compare our results to a canonical tRNA and a mitochondrial tRNA (mtRNA) that lacks the D-stem. Surprisingly, our data show that the global conformation and flexibility of the TLD does not change upon SmpB binding. However, Mg2+ ions induce an 8 A compaction of the TLD.