TRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

Abstract

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.