TY - JOUR
T1 - A Mg2+-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding
AU - Schaak, Janell E.
AU - Yakhnin, Helen
AU - Bevilacqua, Philip C.
AU - Babitzke, Paul
N1 - Funding Information:
The authors thank Alexander Yakhnin for technical assistance, and members of the Babitzke and Bevilacqua labs for helpful comments. This work was supported by National Institutes of Health grant GM52840 to P.B. and National Sciences Foundation CAREER Grant MCB-9984129 to P.C.B.
PY - 2003/9/19
Y1 - 2003/9/19
N2 - Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg2+-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5′-p-azidophenacyl-containing transcripts revealed a Mg 2+-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg2+ inhibits TRAP binding, which may be partially due to the effect of Mg2+ on RNA tertiary structure formation. Results from expression studies using trpE′-′lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg2+-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.
AB - Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg2+-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5′-p-azidophenacyl-containing transcripts revealed a Mg 2+-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg2+ inhibits TRAP binding, which may be partially due to the effect of Mg2+ on RNA tertiary structure formation. Results from expression studies using trpE′-′lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg2+-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.
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U2 - 10.1016/S0022-2836(03)00969-0
DO - 10.1016/S0022-2836(03)00969-0
M3 - Article
C2 - 12963367
AN - SCOPUS:0042736855
SN - 0022-2836
VL - 332
SP - 555
EP - 574
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -