Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus

Abstract

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg 2+ -dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg -and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

The Bacillus subtilis TRAP Protein Can Induce Transcription Termination in the Leader Region of the Tryptophan Biosynthetic (trp) Operon Independent of the trp Attenuator RNA

“…Attempts to map the RNA structure of the trp attenuator, either in the presence of TRAP bound to the leader region RNA or by using mutant leader regions in which the antiterminator is disrupted or deleted, have only provided evidence for base-pairing of the three GC base pairs at the base of the stem (N. Merlino and P. Gollnick unpublished observations). In contrast, the other predicted RNA structures in the trp leader region, including the 59 stem-loop, the antiterminator and the ribosome binding site sequestering structure, have been extensively mapped [34][35][36]. This inability to structure map the attenuator hairpin is surprising given the predicted DG of 212.6 kcal/mol for formation of this structure [37], but is consistent with the weak intrinsic termination activity of the attenuator.…”

Comparative Study between Transcriptionally- and Translationally-Acting Adenine Riboswitches Reveals Key Differences in Riboswitch Regulatory Mechanisms

“…We thus carried out thermal denaturation experiments (TDE) of the pbuE aptamer to determine whether ligand-binding induces aptamer stabilization. TDE monitors the heat-induced unfolding of the RNA as a function of temperature by observing absorbance changes [47] . Using TDE, we followed the absorption of the aptamer at 258 nm in absence and in presence of DAP ( Figure 4D ).…”

Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM

“…For Trp loaded WT-Mut dTRAP, the Cryo-EM structure of holo dTRAP was used as a starting model. The C6 symmetry reconstructed map and initial model were used to refine a single linked dimer using multiple iterations of ISOLDE and Phenix Real Space Refinement [6][7][8] . The last five rounds of iterations were performed using the full dTRAP ring constructed using the apply NCS operators' tool by PHENIX so that the interface between the protomers is properly modeled.…”

“…The RNA structures that are formed upon TRAP binding play roles in down-regulating expression of the Trp biosynthesis machinery by promoting both transcription attenuation and translational repression [1]. Because of these properties TRAP has served as a paradigm for understanding ligand-modulated gene expression [1][2][3], ligand-coupled protein folding [4,5], protein-mediated RNA remodeling [6][7][8], and homotropic and heterotropic cooperativity [9][10][11][12][13][14][15][16], and as a tool for nanotechnology [17,18]. As a highly specific ligand-activated gene regulatory protein [12,[19][20][21], TRAP also has potential as a template for systems biology applications [22].…”