CHARACTERIZATION OF A SINGLE TRAM DOMAIN RNA-BINDING PROTEIN FROM THE ANTARCTIC METHANOGEN METHANOCOCCOIDES BURTONII Taha A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faulty of Science The University of New South Wales March 2016 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: TAHA First name: Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Biotechnology and Biomolecular Sciences Faculty: Faculty of Science Title: Characterization of a single TRAM domain RNA-binding protein from the Antarctic methanogen Methanococcoides burtonii Abstract 350 words maximum: (PLEASE TYPE) TRAM domain proteins present in Archaea and Bacteria have a -barrel shape with anti-parallel -sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins. This thesis explores evolutionary, biophysical, structural and nucleic acid binding properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii. o Ctr3 binds RNA and unfolds reversibly with a two-state mechanism (Tm of ~ 50 C). An on-column in vitro binding assay was used to capture M. burtonii RNA targets of Ctr3. Identification of the captured RNA using RNA-seq revealed that Ctr3 bound M. burtonii RNA with a preference for tRNA and 5S rRNA, and a potential binding motif was identified. In tRNA the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. In 5S rRNA the motif represented one side of the stem and loop C which also appears to be solvent exposed providing possible access to Ctr3. At low temperatures, nucleic acids are prone to form stable secondary structures which consequently impede transcriptional and translational processes in the cell. In Bacteria, a family of Csps has been postulated to resolve inhibitory structures of nucleic acids; thereby facilitating transcription and translation at low temperatures. However, while being ubiquitous in bacterial genomes, only a few csp homologs have been identified in Archaea. The tertiary structures of Ctr3 and Csps are similar. The broad representation of single TRAM domain proteins within Archaea compared to their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA- binding proteins. Although, there is little sequence identity among TRAM domain and Csp proteins, based on evolutionary and tertiary structural analyses, both proteins are inferred to play important roles in cold adaptation, including low temperature translation. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). .. 31/03/2016 .... Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. I ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date ...…………………………………………….............. II Abstract Methanococcoides burtonii, a methanogen that was isolated from Ace Lake, Antarctica, has proven to be a useful model for studying the molecular mechanisms of cold adaptation in Archaea. In Ace Lake, M. burtonii only experiences temperatures below 4oC and nucleic acid binding proteins have been inferred to play important roles in cold adaptation. In this thesis, the three M. burtonii ctr (cold-responsive TRAM domain) genes (proteins) Mbur_0304, Mbur_0604 and Mbur_1445 are referred to as ctr1 (Ctr1), ctr2 (Ctr2) and ctr3 (Ctr3), respectively. The thesis reports the first experimental studies to assess evolutionary, biophysical and structural properties and unique characteristics related to nucleic acid binding, including specific interactions describing putative roles of Ctr3 in the cell. During purification, E. coli nucleic acids consistently co-purified with recombinant Ctr3. The liberated nucleic acid from proteins was able to be digested with RNase indicating the bound nucleic acid was RNA. The bound RNA was able to be removed by treating the recombinant proteins with mild urea to partially unfold the protein, eluting the protein across a NaCl gradient, and refolding it by dialysis. Purification of recombinant RNA-free proteins allowed thorough assessment of the structure-function- stability relationship of Ctr3. Ctr3 unfolded reversibly with a two-state mechanism (Tm of ~ 50oC). The predicted three-dimensional structure of Ctr3 exhibited substantial structural similarities with the TRAM domain of RumA protein (RlmD) from E. coli. The aromatic residues, particularly the four phenylalanine residues of Ctr3 appeared to be surface exposed and in close proximity to each other on the putative RNA-binding surface, similar to the aromatic residues in RumA-TRAM domain, suggesting similar roles in RNA interaction. An on-column in vitro binding assay was used to capture M. burtonii RNA targets of Ctr3, and analysed relative to a complete reconstruction of the M. burtonii transcriptome obtained from total RNA. Identification of the captured RNA using RNA-seq revealed that Ctr3 bound M. burtonii RNA with a preference for tRNA and 5S rRNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. In 5S rRNA, the motif represented one side of the stem and loop C which also appears to be solvent exposed providing possible access to Ctr3. III At low temperatures, nucleic acids are prone to form stable secondary structures which consequently impede transcriptional and translational processes in the cell. In Bacteria, a family of cold shock proteins (Csp) has been postulated to resolve inhibitory structures of nucleic acids; thereby facilitating transcription and translation at low temperatures. However, while being ubiquitous in bacterial genomes, only a few csp homologs have been identified in psychrophilic Archaea; and csp genes are absent from the M. burtonii genome. The first insight into which genes in Archaea may perform an analogous function to csp genes came from proteomic analyses of M. burtonii where the increased abundance of Ctr proteins at low temperature was identified (Williams et al., 2011). The highest levels of these Ctr proteins occurred at 1 and -2°C and in particular, Ctr3 exhibited the highest increases (9-fold) at -2C. Ctr3 and Csps both form a β-barrel shape with anti-parallel -sheets that form a nucleic acid binding surface. The broad representation of single TRAM domain proteins within Archaea compared to their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins. Although, there is little sequence identity among TRAM domain and Csp proteins, based on evolutionary and tertiary structural analyses, both proteins are inferred to play important roles in cold adaptation, including low temperature translation. IV List of Publications Taha., Siddiqui, K.S., Campanaro, S., Najnin, T., Deshpande, N., Williams, T.J., Aldrich‐Wright, J., Wilkins, M., Curmi, P.M.G., and Cavicchioli, R. (2016) Single TRAM domain RNA‐binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii. Environmental Microbiology. doi: 10.1111/1462-2920.13229. Najnin, T., Siddiqui, K.S., Taha., Elkaid, N., Kornfeld, G., Curmi, P.M.G. and Cavicchioli, R. (2016) Temperature-sensing in the Antarctic archaeon, Methanococcoides burtonii: role of a cold-active sensor kinase from a two component regulatory system. Scientific Reports 6:24278 doi:10.1038/srep24278.