TBDB: a database of structurally annotated T-box riboswitch:tRNA pairs

Abstract

T-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-box riboswitches is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches, and de novo prediction of binding specificities requires advanced knowledge of computational tools to annotate riboswitch secondary structure features. Here, we present the T-box Riboswitch Annotation Database (TBDB, https://tbdb.io), an open-access database with a collection of 23,535 T-box riboswitch sequences, spanning the major phyla of 3,632 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory targets. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

Figure 1.

T-box riboswitches are cis-regulatory elements that use tRNA as a ligand. T-box riboswitches control translation or transcription of downstream genes. When uncharged cognate tRNA binds the T-box riboswitch, transcription or translation can proceed through stabilization of the antiterminator/antisequestrator structures. If charged cognate tRNA binds the T-box riboswitch, a terminator/sequestrator secondary structure forms preventing transcription or translation of downstream gene. (A) An archetypal ‘two-state’ conformational switch of a transcriptional T-box riboswitch is shown with structural features labeled: I = Stem I; II = Stem II; IIA/B = Stem IIA/B; III = Stem III; AT = antiterminator; T = terminator; Spec = specifier sequence (blue); T-box = T-box 5′-UGGN-3′ sequence (gray). (B) Watson–Crick base pairing between T-box riboswitch and tRNA in two critical regions dictate T-box riboswitch binding specificity: (i) specifier:anticodon base pairing dictates tRNA specificity while (ii) T-box bulge 5′-UGGN-3′ sequence:tRNA acceptor end base pairing controls regulatory logic.

Figure 2.

Construction of TBDB. T-box riboswitch structures were predicted from input sequences using INFERNAL and RNAfold (Vienna RNA) (35,36). T-box riboswitch features (specifier sequence and T-box riboswitch sequence) were extracted from structural predictions. For input sequences where genomic information was not provided, BLAST (NCBI) was used to identify genetic locus within host. Entrez (NCBI) queries were used to compile all genomic sequence records of the host organisms including genes found downstream of T-box riboswitch input sequences. tRNAscan-SE was run on all genomes to find tRNAs in the hosts with anticodons that are reverse complements of T-box riboswitch specifier sequences (39). Predicted structures were refined using ViennaRNA (36). Refined structures, with predicted features, were visualized as 2D representations using VARNA (40). Minimum free energy (MFE) calculations were performed using ViennaRNA on refined terminator and antiterminator/antisequestrator structures.