PhD Candidate, The University of Chicago
Investigations into enzymatic functions of RNA that were central to the origin of life
Chemistry of Life is written in the alphabet of genes, comprising letters of DNA, arranged in patterns to create biological messages. Sequences of DNA are transcribed into RNA which are used to make proteins. Proteins facilitate biochemical transformations required to sustain life, acting as enzymes or biocatalysts. However, it is generally accepted that RNA carried out the function of both proteins and DNA in their absence in primitive earth. This is the RNA World hypothesis, which proposes that building blocks of RNA, polymerized to form the first RNA molecules, billions of years ago. These molecules assumed different structures, dictated by the sequence of polymerization, and eventually new functions emerged as attributes of particular structures. Emergence of a particular enzymatic function, self-replication, triggered the conversion of non-living matter into life. I study the structure of RNAs that catalyze biochemical reactions, i.e. RNAs that function as enzymes (also Ribozymes). My work has unraveled mechanisms that ribozymes use to carry out their catalytic functions and have yielded useful models for studying the most primitive form of enzyme activity. While it is important to understand the strategies used by RNA enzymes in catalytic function, it is also essential to figure out how these functions emerged. With increasing diversity of RNA-based life, it was necessary for RNA molecules to acquire new functions, which likely occurred through mutations or random changes in its sequence. Novel functions emerged with new sequences adopting distinct structures. As a logical extension of my studies on ribozyme structure and function, the second part of my dissertation investigates the evolutionary mechanisms that could’ve led to the emergence of new enzyme functions in RNA. My research has unveiled evolutionary mechanisms that functional RNAs could’ve used to acquire new functions. Most random mutations create non-functional RNA sequences leading to evolutionary dead ends and therefore are not robust in introducing new functions on their own. I have discovered alternate mechanisms that could compliment mutational changes to explain the emergence of a wide range of enzymatic functions in RNA. My graduate research has uncovered fundamental structural principles guiding RNA enzyme function and delineated molecular mechanisms for the evolution of these functions that played essential roles in the origin of life and sustaining it right after its ‘birth’.
Abstract: The emergence of homeostatic mechanisms that enable maintenance of an intracellular steady state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles, and ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behaviour: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest that such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.
Pub.: 14 Mar '16, Pinned: 29 Jun '17
Abstract: The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
Pub.: 29 Sep '15, Pinned: 29 Jun '17
Abstract: The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme resulting in a rearrangement of the substrate helix register into a so-called 'shifted' conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.
Pub.: 20 Jun '17, Pinned: 29 Jun '17
Abstract: The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.
Pub.: 25 Jun '17, Pinned: 29 Jun '17
Abstract: Either to sustain autotrophy, or as a prelude to heterotrophy, organic synthesis from an environmentally available C1 feedstock molecule is crucial to the origin of life. Recent findings augment key literature results and suggest that hydrogen cyanide—“Blausäure”—was that feedstock.
Pub.: 29 Oct '15, Pinned: 29 Jun '17
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