ORCID Profile
0000-0003-1807-5708
Current Organisation
University of Sydney
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Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Medical Biochemistry and Metabolomics | Medical Biochemistry: Nucleic Acids | Structural Biology (incl. Macromolecular Modelling) |
Publisher: Bentham Science Publishers Ltd.
Date: 28-08-2015
DOI: 10.2174/1389203716666150505230806
Abstract: Cell viability is only possible due to a dynamic range of essential nucleic acid-protein complex formation. DNA replication and repair, gene expression, transcription and protein synthesis are well-known processes mediated by nucleic acids (DNA and RNA) - protein interactions. Novel nucleic acid- protein complexes have been identified in the past few years aided by the development of numerous new techniques such as RNA capture or Tandem RNA Affinity Purification (TRAP). However, the biophysical and biochemical details of these interactions are mostly unknown. Here, we present three techniques (Electrophoretic Mobility Shift Assays, Microscale Thermophoresis and Surface Plasmon Resonance) that are commonly used to quantify and characterize DNA-protein and RNA-protein interactions and discuss their main advantages and limitations.
Publisher: Elsevier BV
Date: 10-2021
Publisher: American Chemical Society (ACS)
Date: 12-2007
DOI: 10.1021/CB7002253
Publisher: Informa UK Limited
Date: 09-2009
DOI: 10.4161/RNA.6.4.9332
Publisher: Frontiers Media SA
Date: 25-05-2021
DOI: 10.3389/FMOLB.2021.679584
Abstract: The signal recognition particle (SRP) is a ribonucleoprotein complex fundamental for co-translational delivery of proteins to their proper membrane localization and secretory pathways. Literature of the past two decades has suggested new roles for in idual SRP components, 7SL RNA and proteins SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72, outside the SRP cycle. These noncanonical functions interconnect SRP with a multitude of cellular and molecular pathways, including virus-host interactions, stress response, transcriptional regulation and modulation of apoptosis in autoimmune diseases. Uncovered novel properties of the SRP components present a new perspective for the mammalian SRP as a biological modulator of multiple cellular processes. As a consequence of these findings, SRP components have been correlated with a growing list of diseases, such as cancer progression, myopathies and bone marrow genetic diseases, suggesting a potential for development of SRP-target therapies of each in idual component. For the first time, here we present the current knowledge on the SRP noncanonical functions and raise the need of a deeper understanding of the molecular interactions between SRP and accessory cellular components. We examine diseases associated with SRP components and discuss the development and feasibility of therapeutics targeting in idual SRP noncanonical functions.
Publisher: Oxford University Press (OUP)
Date: 02-2019
Abstract: The insertion sequence IS26 plays a major role in the mobilization, expression and dissemination of antibiotic resistance genes in Gram-negative bacteria. Though IS26 is abundant in sequenced genomes and in plasmids that harbour antibiotic resistance genes, only a few minor variations in the IS26 sequence have been recorded. The most common variant, IS26* (also known as IS15Δ1), encodes a Tnp26 transposase with a single amino acid substitution, G184N in the catalytic domain. Using computational modelling, this substitution was predicted to increase the length of the helix that includes the E173 residue of the catalytic DDE triad, and its effect on activity was tested. An IS26 mutant generated in vitro producing Tnp26-G184N formed cointegrates in a standard untargeted reaction at 5-fold higher frequency than IS26 producing Tnp26. When the target included a single copy of IS26, the G184N substitution increased the cointegration frequency 10-fold and the reaction was targeted and conservative. Hence, the substitution increased Tnp26 activity. The longer helix may stabilise the position of the E173 of the DDE for the catalysis reaction and the specific G184N substitution may also enhance activity by increasing binding to the terminal inverted repeats.
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH14319
Abstract: RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 11-02-2011
Abstract: The structure provides insight into how protein synthesis is initiated and into the evolution of the eukaryotic ribosome.
Publisher: Frontiers Media SA
Date: 16-08-2022
DOI: 10.3389/FMOLB.2022.975322
Abstract: Long non-coding RNAs (lncRNAs) are RNA transcripts that are over 200 nucleotides and rarely encode proteins or peptides. They regulate gene expression and protein activities and are heavily involved in many cellular processes such as cytokine secretion in respond to viral infection. In severe COVID-19 cases, hyperactivation of the immune system may cause an abnormally sharp increase in pro-inflammatory cytokines, known as cytokine release syndrome (CRS), which leads to severe tissue damage or even organ failure, raising COVID-19 mortality rate. In this review, we assessed the correlation between lncRNAs expression and cytokine release syndrome by comparing lncRNA profiles between COVID-19 patients and health controls, as well as between severe and non-severe cases. We also discussed the role of lncRNAs in CRS contributors and showed that the lncRNA profiles display consistency with patients’ clinic symptoms, thus suggesting the potential of lncRNAs as drug targets or biomarkers in COVID-19 treatment.
Publisher: American Chemical Society (ACS)
Date: 28-02-2006
DOI: 10.1021/BI0523005
Publisher: Wiley
Date: 19-08-2014
DOI: 10.1016/J.FEBSLET.2014.07.039
Abstract: Gene expression is controlled through a complex interplay among mRNAs, non-coding RNAs and RNA-binding proteins (RBPs), which all assemble along with other RNA-associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA-binding domains. However, various methods have been recently developed to capture an RNA of interest and comprehensively identify its associated RBPs. In this review, we discuss the RNA-affinity purification methods followed by mass spectrometry analysis (AP-MS) RBP screening within protein libraries and computational methods that can be used to study the RNA-binding proteome (RBPome).
Publisher: Cold Spring Harbor Laboratory
Date: 28-09-2020
DOI: 10.1101/2020.09.27.315978
Abstract: In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in dissociation from the DNA template. ANTAR domains are a class of antiterminator that bind and stabilise mutually exclusive dual hexaloop motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from E. faecialis, both in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate the conformational changes that occur upon RNA binding and reveal the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.
Publisher: Oxford University Press (OUP)
Date: 12-02-2022
DOI: 10.1093/NAR/GKAC074
Abstract: Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of pathways for overriding termination, one of which is the formation of mutually exclusive RNA motifs. ANTAR domains are a class of antiterminator that bind and stabilize dual hexaloop RNA motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from Enterococcus faecialis, in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate conformational changes that occur upon RNA binding and reveal that the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. An ANTAR domain dimer must contact each hexaloop of the dual hexaloop motif in idually to prevent termination in eubacteria. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.
Publisher: Frontiers Media SA
Date: 05-02-2018
Publisher: American Chemical Society (ACS)
Date: 06-2006
DOI: 10.1021/CB600200K
Abstract: The aminoacyl-tRNA synthetases (aaRSs) are responsible for selecting specific amino acids for protein synthesis, and this essential role in translation has garnered them much attention as targets for novel antimicrobials. Understanding how the aaRSs evolved efficient substrate selection offers a potential route to develop useful inhibitors of microbial protein synthesis. Here, we discuss discrimination of small molecules by aaRSs, and how the evolutionary ergence of these mechanisms offers a means to target inhibitors against these essential microbial enzymes.
Publisher: Portland Press Ltd.
Date: 06-01-2015
DOI: 10.1042/BJ20141140
Abstract: Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a ‘simple’ domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these ‘simple’ SSBs interact with ssDNA.
Publisher: Elsevier BV
Date: 10-2022
DOI: 10.1016/J.PEP.2022.106121
Abstract: The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.
Publisher: EMBO
Date: 08-2005
Publisher: American Association for the Advancement of Science (AAAS)
Date: 18-02-2011
Abstract: Guanine triphosphate controls changes in the signal recognition particle that facilitate transfer of the signal sequence to the translocon.
Publisher: Proceedings of the National Academy of Sciences
Date: 28-08-2017
Abstract: In recent years, noncoding RNA transcripts have been found to interact with genes and modulate their ability to be transcribed and made into protein. Here we uncover many of the mechanistic underpinnings involved in how noncoding RNAs control gene transcription. Notably, we find that noncoding RNA control of gene transcription is based on a combination of structural and sequence components of the noncoding RNA and targeted gene. Collectively, the observations presented here suggest that a much more complex and vibrant RNA regulatory world is operative in gene expression and evolution of the genome.
Publisher: BENTHAM SCIENCE PUBLISHERS
Date: 04-10-2020
Publisher: Elsevier BV
Date: 08-2003
DOI: 10.1016/S1097-2765(03)00280-6
Abstract: Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.
Publisher: Public Library of Science (PLoS)
Date: 25-07-2018
Publisher: Elsevier BV
Date: 04-2004
Publisher: American Chemical Society (ACS)
Date: 27-08-2004
DOI: 10.1021/BI0490542
Abstract: Within the two unrelated aminoacyl-tRNA synthetase classes, lysyl-tRNA synthetase (LysRS) is the only ex le known to exist in both classes. To probe the role of the amino acids responsible for L-lysine binding in the active site of the class II LysRS (LysRS2), we studied the lysS-encoded Escherichia coli protein. On the basis of the structure of L-lysine complexed with E. coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination were systematically replaced. Steady-state kinetic parameters for these variants showed reductions in the catalytic efficiency (k(cat)/K(M)) of 1-3 orders of magnitude, allowing the assignment of specific roles for key residues in the active site of LysRS2. To further investigate the role of each residue in discrimination against noncognate amino acids, steady-state kinetic parameters were determined for the nonprotein amino acid S-(2-aminoethyl)-L-cysteine, a potent inhibitor of LysRS2. While a number of variants showed reductions of several hundred-fold in efficiency of S-(2-aminoethyl)-L-cysteine utilization, this was uniformly accompanied by similar reductions in the efficiency of lysine utilization. Thus, manipulation of the amino acid binding site only allowed up to a 4-fold improvement in S-(2-aminoethyl)-L-cysteine discrimination. This is in contrast to the highly effective discrimination against S-(2-aminoethyl)-L-cysteine by class I LysRS and correlates with the fundamentally different roles of conserved aromatic residues in the two LysRS active sites. This now provides a mechanistic basis for the proposal that differences in amino acid discrimination have been pivotal in the evolution of two unrelated LysRSs.
Publisher: Public Library of Science (PLoS)
Date: 18-07-2012
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.JSB.2019.09.004
Abstract: The bacterial signal recognition particle (SRP) receptor, FtsY, participates with the SRP in co-translation targeting of proteins. Multiple crystal structures of the NG domain of E. coli FtsY
Publisher: Elsevier BV
Date: 12-2013
Location: Brazil
Location: United States of America
Start Date: 01-2013
End Date: 01-2016
Amount: $270,000.00
Funder: Australian Research Council
View Funded Activity