Non-Conserved Residues in Clostridium acetobutylicum tRNA(Ala) Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

Abstract

The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNA(Ala) directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNA(Ala) (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNA(Ala) species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNA(Ala) may have coevolved with the homologous alaS T box leader RNA for efficient antitermination.

Keywords: T box; antitermination; riboswitch; tRNA.

Figure 1

The predicted structural models of alaS T box leader RNAs. (A) C. acetobutylicum alaS. (B) B. subtilis alaS. Numbers start at the predicted transcription start-site (+1) for each gene. The RNAs are shown in the terminator form, and the alternative antiterminator structures are shown to the right of the terminator structures. Conserved sequence and structural elements, including the GA motif, S-turn, AG bulge, Specifier Loop and T box, and the pseudoknot (purple) are labeled. Asterisks indicate highly conserved nucleotides. Bases on the 5’ side of the terminator (blue) pair with part of the conserved T box sequence (red) to form the antiterminator. The GCU alanine Specifier Sequence (boxed) that pairs with the anticodon of tRNA^Ala, and the UGGU residues in the antiterminator bulge that pair with the ACCA acceptor end of tRNA^Ala, are shown in green. Watson-Crick pairs are indicated by “-”, wobble pairs are indicated by “•”.

Figure 2

Cloverleaf diagrams of tRNA^Ala. (A) B. subtilis tRNA^Ala (GGC). (B) B. subtilis tRNA^Ala (UGC). (C) C. acetobutylicum tRNA^Ala (UGC). Sequence differences between the heterologous tRNA^Ala (UGC) molecules ((B) and (C)) are boxed and assigned a letter (A/a, B/b, C/c, D/d and E/e); lower case letters in red represent sequences found in B. subtilis tRNA^Ala (UGC) (abcde), and upper case letters in green represent sequences found in C. acetobutylicum tRNA^Ala (UGC) (ABCDE). tRNA numbers are assigned based on tRNA numbering rules [23].

Figure 3

Antitermination of alaS genes in vitro. (A) B. subtilis alaS. (B) C. acetobutylicum alaS. Transcription was carried out in the presence of 1 or 10 µM T7 RNAP-transcribed tRNA^Ala and percent readthrough (% RT) was corrected for tRNA-independent readthrough. All experiments were repeated at least twice and error bars represent the standard error of the mean (SEM). Bsu, B. subtilis; Cac, C. acetobutylicum.

Figure 4

Antitermination of the C. acetobutylicum alaS gene using C. acetobutylicum and B. subtilis tRNA^Ala (UGC). Single round transcription reactions of the C. acetobutylicum alaS leader template were performed in the presence of increasing amounts of tRNA^Ala (final concentration 0, 0.25, 0.5, 1.0, 5.0, 10 μM). tRNA-independent readthrough was subtracted from each data point, and the resulting values were fit to a hyperbolic equation to determine the K_1/2 and RT_max. All experiments were repeated at least twice and error bars represent the standard error of the mean (SEM). ND (not determined) indicates that the K_1/2 value was too high to be determined.

Figure 5

Effects of tRNA mutations on antitermination. Cloverleaf model of B. subtilis tRNA^Ala (UGC) with sequences shown at five regions (abcde, red) where there are differences from C. acetobutylicum tRNA^Ala. The corresponding sequences from C. acetobutylicum tRNA^Ala (UGC) (ABCDE, green) were introduced as indicated. The resulting constructs were tested for antitermination of the C. acetobutylicum alaS template to determine the RT_max and K_1/2. Asterisk (*) indicates that the RT_max and K_1/2 were calculated without the values determined at the highest two tRNA concentrations. All experiments were repeated at least twice and error bars represent the standard error of the mean (SEM). ND (not determined) indicates that the K_1/2 value was too high to be determined accurately.

Figure 6

Antitermination of C. acetobutylicum alaS leader RNA using tRNA^Ala mutants. Elements ABCDE found in C. acetobutylicum tRNA^Ala (UGC) were sequentially introduced into B. subtilis tRNA^Ala (UGC), and the resulting constructs were tested for C. acetobutylicum alaS antitermination to determine RT_max and K_1/2. ^a: ND not determined. ^b: The K_1/2 and RT_max were calculated without the values of 0.2 and 0.5 µM tRNA.

Figure 7

Antitermination of C. acetobutylicum alaS by tRNA^Ala (UGC). The RT_max and K_1/2 for each construct were determined by in vitro antitermination assays. C. acetobutylicum tRNA^Ala (ABCDE) and B. subtilis tRNA^Ala (abcde) are circled. ND (not determined) indicates that the K_1/2 value was too high to be determined accurately.

Figure 8

Positions of substitutions on tRNA tertiary structure. Yeast tRNA^Phe three-dimensional structure (adapted from [31]) is used to demonstrate the relative positions of elements tested in this study. Anticodon stem-loop (green), D stem-loop, (black), variable loop (orange), T stem-loop (blue), and acceptor arm (red). Locations of substitutions tested in this study are indicated.