In vitro selection to identify determinants in tRNA for Bacillus subtilis tyrS T box antiterminator mRNA binding

Abstract

The T box transcription antitermination regulatory system, found in Gram-positive bacteria, is dependent on a complex set of interactions between uncharged tRNA and the 5'-untranslated mRNA leader region of the regulated gene. One of these interactions involves the base pairing of the acceptor end of cognate tRNA with four bases in a 7 nt bulge of the antiterminator RNA. In vitro selection of randomized tRNA binding to Bacillus subtilis tyrS antiterminator model RNAs was used to determine what, if any, sequence trends there are for binding beyond the known base pair complementarity. The model antiterminator RNAs were selected for the wild-type tertiary fold of tRNA. While there were no obvious sequence correlations between the selected tRNAs, there were correlations between certain tertiary structural elements and binding efficiency to different antiterminator model RNAs. In addition, one antiterminator model selected primarily for a kissing tRNA T loop-antiterminator bulge interaction, while another antiterminator model resulted in no such selection. The selection results indicate that, at the level of tertiary structure, there are ideal matches between tRNAs and antiterminator model RNAs consistent with in vivo observations and that additional recognition features, beyond base pair complementarity, may play a role in the formation of the complex.

Figure 1

(A) Secondary structure model of B.subtilis tyrS leader region and the interaction with tRNA. A Tyr (UAC) specifier sequence in a side bulge of stem–loop I (shown in shadow) base pairs with the anticodon of the cognate tRNA (2,4). Also of importance is the base pairing of the uncharged acceptor end of tRNA with the first four bases (shown in outline) of the antiterminator bulge. This latter interaction also involves covariation of the variable base of the antiterminator (position 222) with the discriminator base of the tRNA (7). (B) Secondary structure of antiterminator model RNA AM1A. The variant model, AM1A(C11U), contains a U at position 11 (corresponding to position 224 in the full leader). These antiterminator model RNAs bind B.subtilis tRNA^Tyr(A73U) in vitro (8) and are functionally relevant in vivo (4). (C) Structure model of B.subtilis tRNA^Tyr(A73U) randomized in D, T and variable loop regions.

Figure 2

(A) Nucleotide sequences of selected clones of randomized tRNA after sixth and seventh in vitro selection cycles against antiterminator model RNA AM1A. Group I corresponds to the kissing tRNA sequences, while Group II corresponds to the functionally relevant tRNA sequences. (B) Nucleotide sequences of selected clones of randomized tRNA after the sixth and seventh in vitro selection cycles against antiterminator model RNA AM1A(C11U). For both (A) and (B), the parentheses next to the sequence number indicate the number of identical sequences. Sequences from randomized regions are shown in capital. Dots indicate the sequences identical to the wild-type B.subtilis tRNA^Tyr(A73U). Lines indicate base deletion. Tertiary base pairing interactions are represented by color coding as shown for the wild-type B.subtilis tRNA^Tyr(A73U) sequence. (See Materials and Methods for evaluation criteria and details regarding selection). Base pair color coding is as follows: 8:14, green; 15:48, red; 18:55, light blue; 19:56, dark blue; 54:58, pink.

Figure 3

Schematic representation of the relative percentage of native tRNA tertiary base pairings observed for each of the selection groups shown in Figure 2. Tertiary base pairings are color coded as in Figure 2. Relative percentage of selected tRNA sequences with designated base pairing represented by line widths as follows: <10%, hairline; 10–19%, 1 pt line; 20–39%, 2 pt line; 39–50%, 4 pt line; >50%, 6 pt line.

Figure 4

Secondary structure of representative selected tRNA/antiterminator complexes involving (A) functionally relevant acceptor end-bulge nucleotide pairing and (B) the kissing tRNA T loop–bulge nucleotide complex. For both, solid lines indicate complementary nucleotides involved in tRNA tertiary structure interactions (for evaluation criteria see Materials and Methods). Dashed lines indicate complementary nucleotides involved in tRNA–antiterminator interaction.