Structural and mechanistic studies of the THI box and SMK box riboswitches.

摘要

Organisms have evolved a variety of mechanisms for regulating gene expression. Expression of individual genes is carefully modulated during different stages of cell development and in response to changing environmental conditions. A number of regulatory mechanisms involve structural elements within messenger RNAs (mRNAs) that, in response to an environmental signal, undergo a conformational change that affects expression of a gene encoded on that mRNA. RNA elements of this type that operate independently of proteins or translating ribosomes are termed riboswitches. In this work, the THI box and SMK box riboswitches were investigated in order to gain insight into the structural basis for ligand recognition and the mechanism of regulation employed by each of these RNAs. Both riboswitches are predicted to regulate at the level of translation initiation using a mechanism in which the Shine-Dalgarno (SD) sequence is occluded in response to ligand binding.;For the THI box riboswitch, the studies presented here demonstrated that 30S ribosomal subunit binding at the SD region decreases in the presence of thiamin pyrophosphate (TPP). Mutation of conserved residues in the ligand binding domain resulted in loss of TPP-dependent repression in vivo . Based on these experiments two classes of mutant phenotypes were identified. Class I mutations resulted in increased accessibility of the SD region and binding of 30S ribosomal subunits regardless of the presence or absence of TPP. In contrast, Class II mutations resulted in constitutive occlusion of the SD region even in the absence of ligand. The latter class represents the first example of riboswitch mutations that result in stabilization of the ligand-bound conformation when no ligand is present.;For the SMK box riboswitch, mutational analysis verified the importance of conserved residues for binding to S-adenosylmethionine (SAM). A minimal SMK box element that retains the ability to bind SAM was used to determine the high resolution structure of the riboswitch in complex with ligand using x-ray crystallography. The SD sequence was shown to be sequestered in the ligand-bound conformation and is essential for SAM binding, providing one of the first examples of a riboswitch in which the regulatory domain is intrinsic to the ligand binding domain. Specific binding to SAM is dictated through direct and indirect contacts with the adenosine and sulfur moieties of the ligand. These results were verified using a fluorescence assay with a variety of SAM analogs. The half-life of the SAM-RNA complex was shown to be much shorter than the half-life of the mRNA inside the cell, suggesting that the RNA may undergo multiple regulatory events in its lifetime. Finally, nuclear magnetic resonance (NMR) was used to investigate the solution structure of the SMK box riboswitch in the presence and absence of ligand. These experiments provided direct evidence of a structural rearrangement of the RNA in response to ligand, consistent with the model and biochemical data.