Factors that influence T box riboswitch efficacy and tRNA affinity

Abstract

The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors.

Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions

“…Transcription termination-readthrough assay was performed essentially as described with modifications 14,25,72 . A hairpin molecular beacon was designed to specifically hybridize to the B. subtilis glyQ downstream readthrough transcript to measure T-box-controlled gene expression (Fig.…”

“…A hairpin molecular beacon was designed to specifically hybridize to the B. subtilis glyQ downstream readthrough transcript to measure T-box-controlled gene expression (Fig. 1a) 72 . It is of the sequence 5'-Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl-3' (Trilink) where upper-and lower-case letters denote deoxy-ribonucleotides and 2'-O-methyl ribonucleotides, respectively 72 Data 4) and linear regression was used to obtain the rate of transcript production in the presence or absence of various tRNAs.…”

“…1a) 72 . It is of the sequence 5'-Cy5-CGCAccuccacuuuucuuucauaaTGCG-Dabcyl-3' (Trilink) where upper-and lower-case letters denote deoxy-ribonucleotides and 2'-O-methyl ribonucleotides, respectively 72 Data 4) and linear regression was used to obtain the rate of transcript production in the presence or absence of various tRNAs. In the presence of robust readthrough transcription, the Square of Pearson Product-Moment Correlation Coefficient (R 2 ) is typically > 0.9.…”

Identification of Spermidine Binding Site in T‐box Riboswitch Antiterminator RNA

“…At higher concentrations of spermidine (>5 m m ), however, destabilization of the antiterminator model RNA occurs as indicated by a switch to decreasing T m and a non‐specific enhancement in inline cleavage across the entire RNA. These results are consistent with polyamine–RNA studies which identified non‐specific destabilization at higher concentrations and are also consistent with the switch to decreased T‐box transcription readthrough efficacy observed at >5 m m spermidine .…”

“…Previous studies with the T‐box riboswitch found that spermidine significantly stimulated transcription readthrough (antitermination) in vitro . This enhanced readthrough efficacy occurred both with and, to a lesser extent, without tRNA, strongly implicating that spermidine might be binding the antiterminator and affecting its function . In this study, we investigate the binding of spermidine to T‐box antiterminator model RNA to determine whether there is a structure‐specific interaction.…”