Characterizing the role of Anti-Trap (AT) in regulating trap-mediated termination of the Bacillus subtilis trp operon
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Attenuation control of the tryptophan ( trp ) EDCFBA operon in the Gram-positive bacterium Bacillus subtilis is mediated by an 11-subunit RNA binding protein called TRAP. When activated by tryptophan, TRAP binds to 11 (G/U)AG repeats in the 5' leader region RNA, and down regulates transcription and translation of the trp biosynthetic genes. An antagonist of TRAP termed Anti-TRAP (AT) is synthesized in response to the accumulation of uncharged tRNA Trp . Prior studies of AT, TRAP and RNA have shown that AT interacts with tryptophan-activated TRAP and prevents it from binding to RNA. However, these studies showed that AT cannot dissociate a pre-formed TRAP-RNA complex. AT forms stable cone-shaped trimers and multiple AT trimers bind to a TRAP 11-mer, but it is not known how many AT trimers must bind to TRAP in order to interfere with binding to RNA. The hypotheses of my Ph.D. work has been to study how AT interacts with TRAP and to study how AT in preventing TRAP-mediated termination during transcription of the trp operon. Our studies show that residues near the top region of the AT trimer are in close proximity to a residue within the RNA binding region of TRAP. Furthermore, we show that AT can prevent TRAP-mediated termination at the trp attenuator during transcription of the leader region as long as TRAP has not bound all 11 (G/U)AG repeats. Under these circumstances, AT functions by inducing TRAP to dissociate from the nascent RNA. Moreover, we demonstrate that 1 AT trimer bound to a TRAP 11-mer reduces the affinity of TRAP to RNA. While approximately 70 species of bacteria contain TRAP, only a few of these contain AT. Prior studies show that expressing RNAs that contain some or all of the (G/U)AGs repeats of a TRAP binding site prevents TRAP-mediated termination at the trp attenuator. Therefore, we hypothesized that bacteria that contain TRAP but lack AT may synthesize non-coding TRAP binding RNAs in response to the accumulation of uncharged tRNA Trp . To test this hypothesis, we performed bioinformatics searches across the genomes of bacterial species that contain TRAP but lack AT, to find regions that might encode non-coding TRAP binding RNAs. Our computational searches so far have not identified good candidates for such non-coding RNAs. However, these studies did identify a novel putative TRAP-binding site overlapping the start codon of a gene encoding S-Adenosylmethionine-dependent methyltransferase (SAM-MTase) in Bacillus clausii . TRAP binding to this target site could potentially regulate translation of this SAM-MTase. We have suggested a model in which this SAM-MTase may play a role in the expression of tRNA synthetases via the methylation of tRNAs, in response to the levels of tryptophan in the cell (see Appendix).