The Role of Base Modifications on Tyrosyl-tRNA Structure, Stability, and Function in Bacillus subtilis and Bacillus anthracis
Nikonowicz, Edward P.
Doctor of Philosophy
tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).