Title

Novel Functions for Large Noncoding Nucleic Acids in Bacteria

Date of Award

Fall 10-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Breaker, Ronald

Abstract

Due to the compact nature of bacterial genomes, large, highly structured noncoding RNAs (ncRNAs) are rare, yet when present these large ncRNAs perform sophisticated biochemical functions, often as ribozymes. Even rarer are DNAs that adapt complex single-stranded structures, as natural opportunities for such structures to form and evolve are limited. Presented in this thesis is research on one large ncRNA and three highly structured, single-stranded DNAs (ssDNAs) that expands our knowledge of the functional capabilities of structured nucleic acids. OLE (Ornate, Large, Extremophilic) RNA is among the largest and most widespread ncRNAs in bacteria without an ascribed biochemical function. The RNA and its protein partner OapA (OLE-associated protein A) are required in Bacillus halodurans for proper adaption to cold temperatures, short chain alcohols, and slightly elevated magnesium. Surprisingly, mutating a conserved DxxxD motif in OapA to AxxxA (named the PM1 strain) causes B. halodurans to become even more sensitive to these stresses but only when OLE RNA is also expressed. This phenotype allowed a genetic screen to reveal additional components to the OLE ribonucleoprotein (RNP) complex, leading to the discovery of YbzG (renamed OapB) as a second OLE RNA-interacting protein and the focus of Chapter 2. OapB is a small (11 kDa), previously uncharacterized protein with a putative RNA binding domain that binds to the P13 region of OLE RNA with subnanomolar affinity (KD ~ 700 pM). This interaction requires the presence of a GNRA tetraloop, though additional contacts are required, as a hairpin with a GNRA tetraloop is insufficient for complex formation. The regions of OLE RNA that OapA and OapB interact with do not overlap, suggesting that the three components can come together to form the OLE RNP complex. I collaborated in an effort led by Dr. Yang Yang to determine the crystal structure of OapB in complex with a subregion of OLE RNA resolved to 2.1 Å, confirming that OapB interacts with the P13 GNRA tetraloop and revealing additional contacts in the P13 and P12.2 stems of OLE RNA. In addition to being found in nearly all ole-containing organisms, OapB is found in 1,670 species that lack the ole gene. To answer the question of what target RNA sites might look like in organisms that lack ole, I generate a consensus model for OapB-RNA interactions through in vitro selection of mutagenized OLE RNA constructs. This model can be used by future researchers to uncover the function of OapB in species that lack ole. In addition to examining the composition of the OLE RNP complex I performed RNA-seq to explore the effect of the OLE RNA on gene expression, highlighted in Chapter 3. These RNA-seq datasets of wild type (WT), ole-oapA, and PM1 strains of B. halodurans grown under either standard or stressed conditions (24 C, 3% EtOH w/v, or 5 mM MgCl2) revealed that multiple metal ion transporters are differentially regulated between WT and ole-oapA or PM1 strains. In addition to my transcriptomics data and the magnesium sensitivity phenotype, bioinformatic evidence also supports the idea that the OLE RNP complex is involved in magnesium homeostasis. The B. halodurans oapA gene is a homolog of the Aeribacillus pallidus citMHS gene, which encodes a magnesium/citrate symporter. If the OLE RNP complex is involved in magnesium homeostasis I hypothesize that one function of OLE RNA may be to act as a fine-tuned sensor for intracellular Mg2+, switching from one conformation to another as Mg2+ concentrations move outside ideal intracellular ranges. RNA is known to undergo structural changes as magnesium concentrations increase, and in the case of magnesium riboswitches those structural changes represent biologically relevant on and off states. I also hypothesize that this conformational switching may regulate OapA. To test this hypothesis, I have conducted in-line probing experiments on OLE RNA at varying Mg2+ concentrations. While my initial results reveal that OLE RNA undergoes a conformation change at biologically relevant Mg2+ concentrations, significant additional work is needed to test whether or not this conformational change is indeed a real regulatory mechanism. Chapter 4 moves away from OLE RNA and focuses on three extraordinarily structured ssDNAs, the HEARO (HNH Endonuclease-Associated RNA and ORF), IS605-orfB-I, and IS605-orfB-II motifs. Each of these motifs is associated with the IS605 superfamily of transposons, a class of mobile genetic elements that transpose via an obligatory ssDNA intermediate. Prior to my work HEARO was believed to function as an RNA. Through bioinformatics, I have established a connection between HEARO and IS605 transposons on both the protein and nucleic acid level, showing that the motif almost certainly functions as an ssDNA. Furthermore, I have determined the phylogenetic distribution and frequency of HEARO elements per genome. For all three motifs I have compared ssDNA structure at the 5’ and 3’ ends of the transposons and analyzed the domain architecture of the TnpB homologs in comparison to canonical IS605 TnpB. Combined, these motifs represent three of the most complex natural ssDNAs and provide powerful insight into the evolution of such motifs.

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