Monitoring Uncharged tRNA During Transcription of the Bacillus subtilis glyQS Gene

Expression of the Bacillus subtilis glyQS gene, encoding glycyl-tRNA synthetase, depends on stabilization of an antiterminator element during transcription of the 5′ region of the mRNA by binding of uncharged tRNA Gly. The glyQS gene is a member of the T box family of genes, all of which are involved in generation of charged tRNA. Each gene in this family exhibits an increase in readthrough of a termination signal located upstream of the start of the coding sequence in response to a decrease in the ratio of charged to uncharged tRNA. Many structural features of T box RNAs that are necessary for tRNA-dependent antitermination have been defined, but little is known about the timing or sequence of events that lead to a productive interaction with uncharged tRNA and discrimination against charged tRNA. To investigate these issues, transcription complexes were blocked artificially at specific positions along the leader sequence and tested for the ability to recognize tRNA. Although the sequence element that binds the tRNA anticodon is located more than 100 nt before the termination signal, complexes with nascent transcripts extending to just upstream of the termination site were still competent for antitermination. This result indicates that the transcript can fold into a receptive structure in the absence of the tRNA, and that tRNA is not necessary prior to this point. A mimic of charged tRNA Gly inhibited antitermination by uncharged tRNA unless the leader RNA–tRNA Gly complexes contained the complete antiterminator. These results suggest that the transcription complex can interact with either uncharged or charged tRNA until it approaches the termination point, allowing maximal flexibility in monitoring the ratio of charged to uncharged tRNA.