NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA

Abstract

Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5' untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have determined the solution structure of the Specifier Loop domain of the tyrS leader RNA from Bacillus subtilis. Fifty percent of the nucleotides in the Specifier Loop domain adopt a loop E motif. The Specifier Sequence nucleotides, which pair with the tRNA anticodon, stack with their Watson-Crick edges rotated toward the minor groove and exhibit only modest flexibility. We also show that a Specifier Loop domain mutation that impairs the function of the B. subtilis glyQS T box RNA disrupts the tyrS loop E motif. Our results suggest a mechanism for tRNA-Specifier Loop binding in which the phosphate backbone kink created by the loop E motif causes the Specifier Sequence bases to rotate toward the minor groove, which increases accessibility for pairing with bases in the anticodon loop of tRNA.

Figure 1.

Sequence and proposed secondary structures of (A) the leader RNA of the B. subtilis tyrS gene and (B) the 38-nt RNA molecule, tyrS_SD, corresponding to the Specifier Loop domain of the tyrS leader RNA.

Figure 2.

(A) Two-dimensional ¹³C-¹H HSQC spectrum of the base C6/8 and C2 regions of tyrS_SD RNA molecule. The sequence-specific resonance assignments are shown. The A₁₃ C2H2 cross-peak is significantly weakened by chemical exchange. The in vitro transcription reaction was primed with unlabeled 5′-GMP, therefore the G₁ C8H8 does not appear in this spectrum. (B) Sequential connectivities through the base-1′ region of the 220-ms mixing time two-dimensional NOE spectrum. The dotted lines trace the connectivities among the specifier codon nucleotides U₂₉–A₃₁. The sequential connectivity is disrupted between steps A₁₀–G₁₁ (gray box). The H1′ resonances of G₁₄ and G₂₂ are shifted upfield to 4.25 and 4.42 p.p.m., respectively. These chemical shifts are characteristic of the nucleotides flanking the 3′ side of the adenine of a sheared G–A base pair and the guanine of a UNCG tetraloop (59). See also Supplementary Table S1.

Figure 3.

(A) Structure of the tyrS_SD RNA with view into the minor groove at the center of the helix. (B) Superposition of 11 lowest energy structures for the full tyrS_SD, and the Specifier Loop domain only. Views are into the minor groove at the center of the Specifier Loop domain. The RMSDs between the individual structures and the average structure are listed in Table 1. The internal loop and stem regions are generally well defined. The disorder of the bulged G₁₁ base reflects the paucity of constraints for this residue.

Figure 4.

Stereoview of the loop E motif bases in the tyrS_SD RNA molecule showing the arrangement of base–base interactions. Hydrogen bonds, supported by ¹⁵N chemical shifts, are indicated by dashed black lines. Hydrogen bonds present in other loop E motifs but for which there is no evidence in the tyrS_SD RNA are shown as dashed red lines. The sheared G–A base pair (pink), reverse Hoogstein A–U base pair (blue), parallel A–A base pair (red) stack on each other. The bulged G₁₁ base (orange) makes no hydrogen bonds with either the phosphate backbone or the flanking bases.

Figure 5.

Stereoview of the minimized average structure of the Specifier Loop domain. The specifier nucleotide bases are colored green and the loop E motif nucleotide bases are colored red. The functional groups on the Watson–Crick edges of the specifier nucleotides are colored pink. The S-turn of the sugar-phosphate backbone can be seen between residues A₈ and G₁₁.

Figure 6.

Overlay of the base C6/8 and C2 regions of ¹³C-¹H HSQC spectra of tyrS_SD RNA molecule (gray) and the tyrS_SD RNA molecule with the substitution U₁₂ to C₁₂ (black). Peaks from the native sequence shifted by the mutation are labeled. Peaks from the sequence containing the U₁₂C mutation that do not overlap peaks from the native sequence are indicated by an arrow. The resonances most affected by the single base substitution correspond to the residues of the loop E motif. Nucleotides in the opposite end of the internal loop, including specifier nucleotides A₃₀ and C₃₁ show much smaller affects.

Figure 7.

Proposed model of the interaction between the Specifier Loop domain and the tRNA anticodon loop. Nucleotide bases in the anticodon loop (red) approach from the minor groove side of the Specifier Domain and form Watson–Crick base pairs with the Specifier Sequence bases (green). Nucleotides of the partner strand (pink) rotate toward the major groove. The S-turn of the phosphate backbone on the partner strand is introduced by the loop E motif (blue) and facilitates the minor groove displacement of the Specifier Sequence bases.