Regulation of gene expression is an essential process that organisms employ for growth and survival. The activation or repression of genes is critical as it allows the cell to monitor environmental signals and adapt to changes in nutrient availability or environmental conditions. One method of regulation involves RNA elements termed riboswitches. Riboswitches are conserved RNA elements that regulate gene expression by modulation of the RNA structure. Typically, a structural change in the RNA occurs in response to environmental signals such as temperature, small RNAs, or small molecules. Most of the known riboswitches modulate gene expression in response to a small molecule effector. Recognition of the effector causes a structural rearrangement that prevents or promotes the formation of a regulatory structure such as an intrinsic transcriptional terminator. Riboswitches that regulate gene expression at the level of translation undergo a structural rearrangement that can occlude or expose the ribosomal binding site (RBS).
In this work, we focused our analysis on the L box riboswitch that regulates gene expression during transcription in Bacillus subtilis and is predicted to regulate at the translational level in Escherichia coli. This conserved RNA regulates the expression of lysine biosynthetic genes in response to cellular lysine concentrations. Previous work revealed that the lysC leader RNA from B. subtilis promotes premature transcription termination in the presence of lysine and can discriminate against lysine analogs. Analysis of the leader region in vivo indicated that lysC expression is repressed when the RNA is transcribed in the presence of high lysine.
Here we investigate the conserved sequence and structural features of the lysC leader riboswitch to elucidate the features that are required for lysine binding and the required structural transition. We have identified variants of the lysC leader RNA that stabilize the termination conformation in the absence of lysine. These results suggest that the energetic balance between the termination and antitermination conformations is sequence-dependent and the conserved nucleotides are critical to the stabilization of the ligand-free structure. In vitro evolution of a randomly mutated lysC leader indicated that regions with conserved structural motifs are also critical to the lysine sensitivity and the structural transition required for lysine-dependent repression. Mutation of non-conserved residues demonstrated that regions distal to the ligand-binding pocket affect ligand sensitivity. Finally, site-directed mutagenesis was used to identify the features of the lysine-binding pocket that are responsible for the lysine specificity. Our results indicate that the majority of the conserved nucleotides within the binding pocket are required for recognition of independent features of the lysine molecule. Mutation of the conserved residues yielded lysC leader variants with alternate ligand specificities. These mutations resulted in variants that are specific for lysine analogs and exhibit a loss of lysine recognition. These experiments provide a comprehensive view of the lysC leader RNA requirements for gene regulation and highlight the conserved sequence and structural features that are necessary for function of the L box riboswitch.
|Commitee:||Alfonzo, Juan, Dean, Don, Foster, Mark|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Gene regulation, L box, Regulatory rna, Riboswitch|
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