Identification of Spermidine Binding Site in T-box Riboswitch Antiterminator RNA.
The T-box transcription antitermination riboswitch controls bacterial gene expression by structurally responding to uncharged, cognate tRNA. Previous studies indicated that cofactors, such as the polyamine spermidine, might serve a specific functional role in enhancing riboswitch efficacy. As riboswitch function depends on key RNA structural changes involving the antiterminator element, the interaction of spermidine with the T-box riboswitch antiterminator element was investigated. Spermidine binds antiterminator model RNA with high affinity (micromolar Kd ) based on isothermal titration calorimetry and fluorescence-monitored binding assays. NMR titration studies, molecular modeling, and inline and enzymatic probing studies indicate that spermidine binds at the 3' portion of the highly conserved seven-nucleotide bulge in the antiterminator. Together, these results support the conclusion that spermidine binds the T-box antiterminator RNA preferentially in a location important for antiterminator function. The implications of these findings are significant both for better understanding of the T-box riboswitch mechanism and for antiterminator-targeted drug discovery efforts.
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Jia
J
Liu
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Chunxi
C
Zeng
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Vivian
V
Hogan
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Shu
S
Zhou
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Md Masud
MM
Monwar
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Jennifer V
JV
Hines
D020029:Base Pairing; D001665:Binding Sites; D018388:Codon, Terminator; D062105:Molecular Docking Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D058928:Riboswitch / Q000502:physiology*; D013095:Spermidine / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D018388:Codon, Terminator; D058928:Riboswitch; D012343:RNA, Transfer; D013095:Spermidine
RNA; T-box; antiterminator; binding; riboswitch; spermidine