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, 290 (38), 23336-47

Codon-Anticodon Recognition in the Bacillus Subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME

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Codon-Anticodon Recognition in the Bacillus Subtilis glyQS T Box Riboswitch: RNA-DEPENDENT CODON SELECTION OUTSIDE THE RIBOSOME

Enrico Caserta et al. J Biol Chem.

Abstract

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the "Specifier Sequence," in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA(Gly) anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3' of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system.

Keywords: RNA synthesis; antitermination; bacterial transcription; ribosome; riboswitch; transcription regulation; transfer RNA (tRNA).

Figures

FIGURE 1.
FIGURE 1.
Models of the B. subtilis glyQS leader RNA and tRNAGly. A, glyQS leader RNA. Conserved structural elements in the glyQS leader RNA are labeled; the Stem II and IIA/B elements are missing in this RNA. The GGC glycine Specifier Sequence (green, boxed) located at positions 99–101 and A102 were mutated to all possible nucleotides (N = G, A, U, or C). The conserved T box sequence is labeled in red, and the bases that pair with the acceptor end of tRNA are labeled in green. The nucleotides that pair with the T box sequence in the antiterminator are labeled in blue. Asterisks indicate nucleotides that are conserved among T box leader RNAs. B, tRNAGly. The GCC anticodon (boxed) corresponding to positions 34–36 and the nucleotide at position 33 were individually mutated to all possible nucleotides. Watson-Crick pairs are shown by a “-,” and non-W-C pairs are shown by “•.”
FIGURE 2.
FIGURE 2.
Effect of pairing alterations at positions 1, 2, and 3 of the Specifier Sequence on tRNA binding and tRNA-dependent transcription antitermination. A, tRNA binding was measured by incubation of increasing concentrations of wild-type glyQS leader RNA with a constant concentration of [32P]tRNA (wild type or mutant) and filtered through 30,000 molecular weight cutoff Nanosep filters to determine the amount of the radiolabeled tRNA retained. The values were used to calculate the dissociation constant (Kd). B, antitermination was measured by incubation of increasing concentrations of tRNAGly and a constant concentration of the wild-type glyQS leader template and determination of the percent readthrough. The values were used to calculate the K1/2. Error bars represent S.E. C, the reduction in binding affinity in the presence of pairing alterations at positions 1, 2, and 3 of the Specifier Sequence was calculated by determining the Kd of the mutant construct relative to the Kd of the wild-type interaction. D, the reduction in antitermination efficiency in the presence of pairing alterations at positions 1, 2, and 3 of the Specifier Sequence was calculated by determining the K1/2 of the mutant variant relative to the K1/2 of the wild-type construct. If the reduction in antitermination efficiency was >20-fold only up to 20 is shown. Detailed values are listed in supplemental Table S1.
FIGURE 3.
FIGURE 3.
Reduction in tRNA binding affinity and antitermination efficiency relative to the estimated stability of an anticodon-anticodon duplex. Pairing interactions at position 1 (A and D), 2 (B and E), or 3 (C and F) of the Specifier Sequence are shown. The Pearson's correlation coefficient (r) was calculated in D excluding the construct that forms a U·C mismatch at position 1 of the Specifier Sequence. Detailed values are listed in supplemental Table S1.
FIGURE 4.
FIGURE 4.
Effect of base alterations at the extended position (position 102 of the glyQS leader RNA and position 33 of tRNAGly). Reductions in tRNA binding affinity (black bars) and antitermination efficiency (white bars) are relative to the wild-type A-U pair. If the reduction in antitermination efficiency was >20-fold only up to 20 is shown. Detailed values are listed in supplemental Table S1.
FIGURE 5.
FIGURE 5.
Comparison of the effects of pairing alterations on antitermination efficiency and binding affinity. Values below 1.0 indicate that a pairing interaction caused a greater defect in tRNA binding affinity than antitermination efficiency; values above 1.0 indicate that a pairing interaction caused a greater defect in antitermination efficiency than in tRNA binding affinity. Detailed values are listed in supplemental Table S1.

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