Poster abstracts
Poster number 116 submitted by Courtney Szyjka
Suppression screen for the identification of protein interactors of the trp RNA-binding attenuation protein (TRAP)
Courtney Szyjka (Department of Biological Sciences, University at Buffalo), Natalie McAdams (Department of Microbiology and Immunology, University at Buffalo), Emily Bonacquisti (Department of Biological Sciences, University at Buffalo), Justin Durland (Department of Biological Sciences, University at Buffalo), Paul Gollnick (Department of Biological Sciences, University at Buffalo)
Abstract:
Transcriptional regulation of the tryptophan (trp) biosynthetic operon in Bacillus subtilis is controlled by the trp RNA-binding attenuator protein (TRAP). Regulation of this operon was initially described as depending on two competing RNA structures present in the leader region upstream of the first gene in the operon. Formation of these structures, designated as the anti-terminator and terminator, is mutually exclusive due to shared bases. In the presence of excess tryptophan, TRAP binds to 11 (G/U)AG repeats in the trp leader region RNA and prevents anti-terminator formation, allowing formation of the terminator thus halting transcription of the trp genes. In limiting tryptophan conditions, TRAP does not bind the trp leader RNA, the anti-terminator forms and the genes are transcribed and translated to produce the tryptophan biosynthesis enzymes. However, recent work has shown that, 1) formation of the terminator RNA structure isn’t sufficient to induce transcription termination in the absence of TRAP and 2) in vivo TRAP can induce termination in the absence of the terminator RNA structure. Together these observations suggest that TRAP may have a more direct role in transcription attenuation. Consistent with this suggestion, we have recently isolated a TRAP mutant (E60K) that is capable of binding RNA and tryptophan at wild-type (WT) levels, but is deficient in transcription attenuation. We used a suppression screen to identify protein interactors of TRAP that are necessary for efficient transcription attenuation in the trp operon. By placing the toxic mazF gene under control of the trp leader region, we identified B. subtilis proteins that will enhance transcription attenuation of the E60K TRAP mutant in vivo. A candidate gene produced from the screen is yteA, a currently uncharacterized gene that shares homology with E. coli transcription factor dksA. Through use of strains with transcriptional fusions between the trp leader and β-galactosidase, we show that a mutant attenuator (disrupted stem) shows decreased TRAP-mediated regulation when yteA is knocked out.
Keywords: transcription attenuation, transcription termination, suppression screen