Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Breaker, Ronald

Abstract

Prior to the 1980s, RNA macromolecules were largely viewed as a transient carrier of the genetic code used for protein synthesis, with little chemical versatility beyond Watson-Crick-Franklin base-pairing. The discovery of catalytic RNAs in 1982, however, upended this view and established that RNA was a chemically capable biopolymer. Since then, several classes of RNA with functions independent from protein synthesis have been discovered in all domains of life. Excluding those that have functions relevant to protein translation, noncoding RNAs that are large (> 200 nt) and have a conserved, complex structure are rare in bacteria. However, as more bacteria were sequenced, comparative sequence analyses revealed the existence of additional, albeit rare, classes of large and highly structured noncoding RNAs. The work presented in this Thesis seeks to further our understanding of the largest, most structurally complex, and most abundant RNA in bacteria whose function remains undefined: the Ornate, Large, Extremophilic (OLE) class of noncoding RNA. In Chapter I, I provide an overview of the major findings from work on OLE RNA that has accumulated over the past 20 years. In brief, members of the OLE noncoding RNA class are typically around 630 nucleotides in length and have a remarkably conserved secondary structure. OLE RNA has been determined to form an RNA dimer that further binds at least four proteins – OapA, OapB, OapC, and ribosomal protein bS21 – to form a large ribonucleoprotein (RNP) complex that resides at bacterial membranes. The biochemical function of the OLE RNP complex, while yet elusive, has been determined to be important for Halalkalibacterium halodurans (formerly called Bacillus halodurans), a model haloalkaliphile, to tolerate a multitude of stress conditions that include cold, short-chain alcohols, moderately excessive Mg2+ concentrations, or growth in minimal media with non-glucose carbon sources. In Chapter II, we present evidence that a protein called YbxF, renamed to OapC, is an essential component of the OLE RNP complex. OapC is a homolog of ribosomal protein L7Ae that binds to and stabilizes kink turn motifs in RNA. This interaction was uncovered using an RNA ‘pulldown’ strategy termed Capture Hybridization Analysis of RNA Targets (CHART) that entailed chemically crosslinking H. halodurans cells with formaldehyde and subsequently purifying OLE RNP complexes from cell lysate. Notably, the CHART experiments further revealed that several more proteins interact with OLE RNA in H. halodurans. In Chapter III, my colleagues and I determined that OLE RNP complex function is critical for H. halodurans growth in minimal media having non-glucose carbon sources. This study was motivated by the observation that a few proteins determined to interact with OLE RNA from Chapter I have biochemical functions relevant to carbon metabolism. To better understand the basis for this growth impairment phenotype, genetic suppressor selections were then performed that ultimately revealed H. halodurans ?ole-oapA cells spontaneously acquire gain-of-function mutations in genes that encode proteins relevant to Mn2+ homeostasis that suppress the suboptimal carbon source growth impairment phenotype. It was then determined that when H. halodurans cells with disrupted OLE RNP complexes are starved for Mn2+ when grown under Mg2+ and carbon source stresses. In Chapter IV, we begin by discussing the incidental finding that H. halodurans ?ole-oapA cells have an increased level of tolerance towards sublethal concentrations of antibiotics that cause ribosomes to translate slowly or stall. We then implemented ribosome-stalling reporter assays and determined that ribosomes in H. halodurans ?ole-oapA cells stall more frequently in the WT strain. Subsequent experiments then determined that ribosome stalling in ?ole-oapA cells correlates with intracellular Mg2+ stress, both of which can be suppressed by supplementing Mn2+ to the growth medium. Lastly, we determined that a knockout strain of mpfA, which encodes a putative Mg2+ exporter, from B. subtilis phenocopies H. halodurans ?ole-oapA with respect to ribosome-targeting antibiotic tolerance and ribosome stalling levels. Chapter V seeks to answer the question: what device(s) do organisms that lack the ole gene use instead to resist ?ole-associated stress conditions? A growing body of literature, including the results presented in Chapter IV, suggested that members from the MpfA protein family may be functionally equivalent to the OLE RNP complex. Experiments expressing mpfA genes from different organisms in H. halodurans ?ole-oapA under Mg2+ stress revealed that all MpfA protein sequences tested conferred the strain resistance to Mg2+, but some sequences displayed a pH dependence, whereas others did not. Subsequent bioinformatic investigations suggested that the MpfA protein family can be divided into subclasses, which align with the observed pH dependence. However, no MpfA protein sequence tested was able to rescue H. halodurans ?ole-oapA from other ?ole-associated stresses except for MpfB from Staphylococcus aureus, which also conferred H. halodurans ?ole-oapA some resistance to cold stress. I then go on to demonstrate that expression of ole and oapA in a B. subtilis strain having knocked out two mpfA genes rescued growth of the strain from Mg2+ stress and conferred the strain some resistance to cold and ethanol stresses. Taken together, these results solidify that the OLE RNP complex likely has a direct role in metal ion homeostasis and support the hypothesis that the MpfA proteins have likely replaced the OLE RNP complex in most other organisms. In Chapter VI, we provide an analysis of the recently determined cryo-EM structures of OLE RNA and discuss the possibility that OLE RNA dimers penetrate bacterial membranes. We conclude this perspective with a discussion of major questions that remain.

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