Uncovering how bacteria sense and respond to chemically diverse corrinoids through cobalamin riboswitch gene regulation

Cells actively sense and integrate information about their internal and external environments to execute adaptive physiological responses. This makes it possible to survive fluctuating and oftentimes harsh environmental conditions. Controlling the expression of genes is part of this vital cellular process. Indeed, since the birth of molecular biology theory, a multitude of cellular mechanisms have been found to control virtually every aspect of the flow of genetic information from DNA to RNA to functional proteins. The revelation that RNAs are more than just passive messengers between DNA and proteins was a turning point in the field of molecular biology. We now recognize that many types of RNA actively fold into complex three-dimensional structures to carry out important cellular functions with sophistication, precision, and efficiency that rivals proteins. A prime example are riboswitches – structured noncoding RNAs that sense specific small molecule effectors by direct binding and function as cis-regulatory genetic switches. My dissertation research focuses on the forms and functions of the cobalamin riboswitches and how they are impacted by the exceptional chemical diversity of naturally occurring variants of cobalamin known as corrinoids./nI begin the first chapter with a broad overview of bacterial gene regulation with specific emphasis on mechanisms of transcriptional and translational control. This sets the stage for delving into the distinctive elements of riboswitch structure, mechanism, and function. I also describe how various riboswitch classes connect to the cellular processes which they control. Lastly, I describe in detail the cobalamin riboswitch class and the roles that corrinoids play in bacterial physiology./nThe second chapter describes the bulk of my endeavors as a graduate student researcher in the Taga Lab examining the corrinoid specificity of cobalamin riboswitches. The large number of bacterial metabolic pathways that involve corrinoids and the numerous types of corrinoid cofactors and intermediates present a puzzle as to how cells can effectively use cobalamin riboswitches to control their corrinoid-related physiology. The approach I took leveraged two strengths of the Taga Lab’s expertise: bacterial molecular genetics and biochemical production of commercially unavailable corrinoid molecules. I engineered an in vivo fluorescence reporter system to measure the responses of several cobalamin riboswitches to several corrinoids. From the patterns of corrinoid selectivity that I observed in my experiments, I developed a mechanistic hypothesis for corrinoid specificity of cobalamin riboswitch-based gene regulation in bacteria. Furthermore, I propose a regulatory strategy that attempts to explain how corrinoid specificities of gene regulation and bacterial physiology are functionally connected./nIn the third chapter, I explore the functional versatility of cobalamin riboswitches. As the second most prevalent class of riboswitches, I speculated that novel functional variations should arise from the diversity of cobalamin riboswitch sequences. To increase the likelihood of finding uncommon functional variants, I focused on atypical cobalamin riboswitch regulon architectures among bacterial species that specialize in corrinoid metabolism. This rationale enabled me to successfully identify a novel activator cobalamin riboswitch, dissect modular functionalities of tandemly linked cobalamin riboswitches, and develop new hypotheses about corrinoid-specific physiology./nTogether, my research studies constitute a step towards reconciling the apparent oversimplicity of current cobalamin riboswitch models with the intrinsic complexity of the cellular processes they control.

Advisor: Taga, Michiko E