Exoribonucleases are indispensable for cellular RNA metabolism. RNA processing, end-turnover, and degradation all require the concerted action of exoribonucleases. In this thesis, two families of exoribonucleases that act in the final steps of RNA decay pathways are explored. The first of these is the RNR superfamily of processive 3’→5’ RNases with major roles in both mRNA and stable RNA degradation. The initial focus of this work is the structural and enzymatic characterization of an unusual RNR family enzyme from the radiation-resistant bacterium Deinococcus radiodurans. This enzyme is demonstrated biochemically to be an RNase II-type enzyme (DrII), based on its sensitivity to secondary structure. Analysis of the DrII X-ray structure reveals that a novel, winged-HTH domain has replaced the canonical RNA binding clamp typical of RNR family proteins. The exposed architecture of DrII’s RNA binding surface offers an explanation for the nuclease’s ability to approach within 3-5 nt of a duplex, an important mechanistic difference from the well-studied E. coli RNase II. The open, clamp architecture of DrII may have broader relevance to mechanisms of duplex RNA recognition in the RNR superfamily. RNA decay by processive exonucleases such as RNR family proteins leaves 2-5 nt nanoRNA limit products that are further degraded to mononucleotides by nanoRNases. In E. coli, the DEDD family enzyme Oligoribonuclease (ORN) executes nanoRNA decay and represents the first major family of nanoRNases, with homologs widely conserved in eubacteria and eukaryotes. The B. subtilis NanoRNase A (NrnA), a DHH family phosphoesterase, represents a second major class of nanoRNases, with broad phylogenetic distribution in organisms that lack orn homologs. The second major focus of this thesis is a structural and mechanistic study of this nanoRNase machinery. The atomic structure of the B. subtillis nanoRNase NrnA is described, and unveils a bi-lobal architecture similar to the 5’→3’ DNase RecJ, where the catalytic DHH domain is linked via a partially helical connector to the C-terminal RNA binding domain. NrnA is a highly dynamic molecule, adopting both open and closed conformations. Co-crystallization with several substrates shows that NrnA has a nanoRNA specific substrate-binding patch that offers a structural explanation for its 3’→5’ nanoRNase activity. This RNA binding site feeds substrate to the DHH active site in an orientation opposite to the 5’→3’ path proposed for RecJ. Surprisingly, NrnA also maintains a weak 5’→3’ activity on certain substrates, and thus possesses both 5’→3’ and 3’→5’ exonuclease activities. In conclusion, an overall model is presented for how DHH family exonucleaess can degrade nucleic acids from both the 5’→3’ and 3’→5’ directions. Thus, the studies described in this thesis offer both an atomic and a biochemical view of the macromolecular machinery critical to the degradation of RNA.

Like all phages, T4 requires a holin (T) to effect lysis. The lysis event depends on the temporally regulated action of T, which accumulates in the inner membrane (IM) until, at an allele-specific time, it triggers to form a large "hole" in the membrane. Hole formation then releases T4 lysozyme into the periplasm where it degrades the cell wall to elicit cell lysis. Unlike other phages, T4 is unique in exhibiting real-time regulation of lysis based on environmental conditions. Specifically, lysis can be delayed indefinitely in the lysis-inhibited state (LIN), where the normal temporal schedule for holin-triggering is over-ridden. Recently, it was shown that the imposition of LIN was correlated with the interaction of the periplasmic domains (PD) of RI and T. These studies have been extended in this dissertation using genetic, biochemical, and structural techniques to address the molecular mechanism of the RI-T LIN system. First, the PD of RI and an RI-T complex were purified, characterized biophysically, and crystallized to yield the first atomic resolution structures of either a holin or antiholin. The RI PD is mostly alpha-helical that undergoes a conformational change, as revealed by NMR spectroscopy studies, when bound to T. The PD of T is globular with alpha-helical, beta strand, and random coil secondary structures. Additionally, the holin was genetically characterized by mutagenesis techniques, yielding new information on its role in both lysis and LIN. Lysis defective mutants in all three topological domains: cytoplasmic, transmembrane, and periplasmic, were isolated. Analysis of these mutants revealed that both the cytoplasmic and periplasmic domains are important in the oligomerization of T. During LIN, the RI PD binds the PD of T, blocking a holin oligomerization interface. Finally, the signal for the imposition of lysis inhibition has been elucidated using NMR spectroscopy and other in vitro studies. These studies have shown that the RI PD binds DNA. From these studies, new models for lysis and LIN have been constructed. Lysis occurs with the accumulation and oligomerization of T via cytoplasmic and periplasmic domain interactions. LIN is imposed when the ectopically localized DNA of a superinfecting phage interacts with RI, stabilizing it in a conformation competent in inhibiting T oligomerization and leading to lysis inhibition.

Thesis (Ph.D.)--McMaster University (Canada), 1994. / Source: Dissertation Abstracts International, Volume: 56-08, Section: B, page: 4314. Adviser: R. F. Childs.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2005. / Wartell, Roger, Committee Member ; Wilkinson, Angus, Committee Member ; Doyle, Donald, Committee Member ; Hud, Nicholas, Committee Member ; Williams, Loren, Committee Chair. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 191-197).

Thesis (Ph. D.)--University of Oregon, 2000. / Title from title screen. Paging within document: xii, 211 p. : ill. (some col.). Includes vita and abstract. Includes bibliographical references (p. 199-211).

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Dr. Allen M. Orville, Committee Chair ; Dr. Loren D. Williams, Committee Member ; Dr. Dale E. Edmondson, Committee Member ; Dr. Frank E. Lffler, Committee Member ; Dr. Nicholas V. Hud, Committee Member

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (M.S.)--Bioengineering, Georgia Institute of Technology, 2007. / Zhu, Cheng, Committee Chair ; Selvaraj, Periasamy, Committee Member ; Orville, Allen, Committee Member ; Butera, Robert, Committee Member.