Protein-RNA interactions involved in regulation of the tryptophan biosynthesis operon (trpEDCFBA) in Bacillus subtilis

Open Access
McGraw, Adam Philip
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
September 24, 2008
Committee Members:
  • Paul Lee Babitzke, Committee Chair
  • Philip C. Bevilacqua, Committee Chair
  • Kenneth Charles Keiler, Committee Member
  • Christine Dolan Keating, Committee Member
  • protein-RNA interactions
  • mRNA stability
  • TRAP
  • RNA structure
Virtually all of RNA’s cellular functions are carried out in concert with proteins. As a result, the complex array of protein-RNA interactions is the driving force behind many cellular activities. In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthesis genes by interacting with the corresponding transcripts. Six of these genes lie in the trpEDCFBA operon, which is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Binding of TRAP blocks formation of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin that prematurely halts transcription, thereby reducing expression of the downstream trp genes. The work presented here shows that a 5’ stem-loop (5’SL) structure that forms just upstream of the triplet repeat region contributes to TRAP-trp RNA interaction by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. This increased affinity allows TRAP to bind shorter transcripts, thereby increasing the efficiency of the transcription attenuation mechanism. Since there is only a short window of time in which binding of TRAP can promote termination, the contribution of the 5’SL to TRAP binding is critical for proper attenuation control. A combination of footprinting assays, affinity assays, phylogenetic comparisons, and mutational studies examining various 5’SL mutants indicate that the 5’SL contacts TRAP through as many as five single-stranded nucleotides that lie within two discrete groups of the RNA structure, the internal loop and the hairpin loop. The intramolecular distance between these two loops is critical to preserving TRAP-5’SL interaction. Results from photochemical crosslinking experiments also show that during TRAP-5’SL interaction, the hairpin loop is in close proximity to the flexible loop region of the protein. These data, combined with molecular modeling of B. subtilis TRAP and of a 3-dimensional model of the 5’SL generated using the MC-Sym and MC-Fold pipeline, have afforded the first molecular-level model of TRAP-5’SL interaction. In addition to participating in TRAP binding, the trp leader 5’SL also participates in turnover of trp mRNA by destabilizing the downstream mRNA sequence, an effect that is opposite to that of most all other bacterial 5’-terminal RNA secondary structures. Mutations altering the 5’SL structure or sequence also led to an increase in mRNA half-life, indicating that the mechanism of 5’SL-mediated destabilization is highly specific. Similarly, chimeric transcripts containing the trp leader 5’SL fused to an unrelated downstream gene showed that the 5’SL alone is insufficient to induce destabilization, suggesting that other components of the trp leader may be necessary to produce this effect. Even though it is involved in the initial stages of the trp transcript decay pathway, it is likely that RNase J1 is not responsible for 5’SL-mediated destabilization of the trp transcript, and further experimentation will be required to identify the associated decay factor. Since some of the 5’SL nucleotides involved in mRNA destabilization also function in TRAP recognition, it appears that two independent regulatory mechanisms have evolved to recognize the same features of an RNA structure, providing an interesting example of the complexity of gene regulation.