Publication Date

2012-03-03

Availability

Embargoed

Embargo Period

2013-03-03

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2012-02-10

First Committee Member

Arun Malhotra

Second Committee Member

Murray P. Deutscher

Third Committee Member

Antonio Barrientos

Fourth Committee Member

Thomas K. Harris

Fifth Committee Member

Bryce Nickels

Abstract

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.

Keywords

NanoRNA; nanoRNases; RNA degradation; Exonucleases; Bacteria; X-ray crystallography

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