Heather C O'Farrell

Virginia Commonwealth University, Richmond, VA, United States

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Publications (9)51.05 Total impact

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    ABSTRACT: The gene orfX is conserved among all staphylococci and its complete sequence is maintained upon insertion of the genomic island SCCmec, containing the gene encoding resistance to beta-lactam antibiotics (mecA), into its carboxy-terminus. The function of OrfX has not been determined. We showed that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth and that the insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belonged to the S-adenosyl methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the E. coli 70S ribosomal MTase rlmH. MTase activity was confirmed by demonstrating the OrfX dependent methylation of the Staphylococcus aureus 70S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, it was found that each monomer of the homodimeric structure binds AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (Kd values of 52 ±0.4 μM and of 606 ±2 μM). In addition, the structure showed that the AdoMet binding pocket, formed by a deep trefoil knot, contained a bound phosphate molecule, which was the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase, and is the first crystal structure of a member of the α/β- knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.
    Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
  • Daniel Boehringer, Heather C O'Farrell, Jason P Rife, Nenad Ban
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    ABSTRACT: The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.
    Journal of Biological Chemistry 02/2012; 287(13):10453-9. · 4.65 Impact Factor
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    ABSTRACT: The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.
    Biochemistry 12/2011; 51(1):466-74. · 3.38 Impact Factor
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    ABSTRACT: The KsgA/Dim1 family of proteins is intimately involved in ribosome biogenesis in all organisms. These enzymes share the common function of dimethylating two adenosine residues near the 3'-OH end of the small subunit rRNA; orthologs in the three kingdoms, along with eukaryotic organelles, have evolved additional functions in rRNA processing, ribosome assembly, and, surprisingly, transcription in mitochondria. The methyltransferase reaction is intriguingly elaborate. The enzymes can bind to naked small subunit rRNA but cannot methylate their target bases until a subset of ribosomal proteins have bound and the nascent subunit has reached a certain level of maturity. Once this threshold is reached, the enzyme must stabilize two adenosines into the active site at separate times and two methyl groups must be transferred to each adenosine, with concomitant exchanges of the product S-adenosyl-l-homocysteine and the methyl donor substrate S-adenosyl-l-methionine. A detailed molecular understanding of this mechanism is currently lacking. Structural analysis of the interactions between the enzyme and substrate will aid in this understanding. Here we present the structure of KsgA from Methanocaldococcus jannaschii in complex with several ligands, including the first structure of S-adenosyl-l-methionine bound to a KsgA/Dim1 enzyme in a catalytically productive way. We also discuss the inability thus far to determine a structure of a target adenosine bound in its active site.
    Biochemistry 02/2010; 49(12):2697-704. · 3.38 Impact Factor
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    Heather C O'Farrell, Zhili Xu, Gloria M Culver, Jason P Rife
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    ABSTRACT: One of the 60 or so genes conserved in all domains of life is the ksgA/dim1 orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. For example, in S. cerevisiae Dim1 performs two distinct functions in ribosome biogenesis, while human mtTFB is not only an rRNA methyltransferase in the mitochondria but also a mitochondrial transcription factor. Thus, these proteins offer an unprecedented opportunity to study evolutionary aspects of structure/function relationships, especially with respect to our recently published work on the binding mode of a KsgA family member to its 30S subunit substrate. Here we compare and contrast KsgA orthologs from bacteria, eukaryotes, and mitochondria as well as the paralogous ErmC enzyme. By using structure and sequence comparisons in concert with a unified ribosome binding model, we have identified regions of the orthologs that are likely related to gains of function beyond the common methyltransferase function. There are core regions common to the entire enzyme class that are associated with ribosome binding, an event required in rRNA methylation activity, and regions that are conserved in subgroups that are presumably related to non-methyltransferase functions. The ancient protein KsgA/Dim1 has adapted to cellular roles beyond that of merely an rRNA methyltransferase. These results provide a structural foundation for analysis of multiple aspects of ribosome biogenesis and mitochondrial transcription.
    BMC Research Notes 11/2008; 1:108.
  • Zhili Xu, Heather C O'Farrell, Jason P Rife, Gloria M Culver
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    ABSTRACT: In contrast to the diversity of most ribosomal RNA modification patterns and systems, the KsgA methyltransferase family seems to be nearly universally conserved along with the modifications it catalyzes. Our data reveal that KsgA interacts with small ribosomal subunits near functional sites, including Initiation factor 3 and 50S subunit binding sites. These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation.
    Nature Structural & Molecular Biology 06/2008; 15(5):534-6. · 11.90 Impact Factor
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    ABSTRACT: Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution and therefore were present in the last common ancestor. In this paper we report that KsgA orthologs from archaeabacteria and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life.
    RNA 06/2006; 12(5):725-33. · 5.09 Impact Factor
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    Heather C O'Farrell, J Neel Scarsdale, Jason P Rife
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    ABSTRACT: The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosyl-l-methionine (S-AdoMet) to two adjacent adenosine bases in 16S rRNA. This enzyme and the resulting modified adenosine bases appear to be conserved in all species of eubacteria, eukaryotes, and archaebacteria, and in eukaryotic organelles. Bacterial resistance to the aminoglycoside antibiotic kasugamycin involves inactivation of KsgA and resulting loss of the dimethylations, with modest consequences to the overall fitness of the organism. In contrast, the yeast ortholog, Dim1, is essential. In yeast, and presumably in other eukaryotes, the enzyme performs a vital role in pre-rRNA processing in addition to its methylating activity. Another ortholog has been discovered recently, h-mtTFB in human mitochondria, which has a second function; this enzyme is a nuclear-encoded mitochondrial transcription factor. The KsgA enzymes are homologous to another family of RNA methyltransferases, the Erm enzymes, which methylate a single adenosine base in 23S rRNA and confer resistance to the MLS-B group of antibiotics. Despite their sequence similarity, the two enzyme families have strikingly different levels of regulation that remain to be elucidated. We have crystallized KsgA from Escherichia coli and solved its structure to a resolution of 2.1A. The structure bears a strong similarity to the crystal structure of ErmC' from Bacillus stearothermophilus and a lesser similarity to sc-mtTFB, the Saccharomyces cerevisiae version of h-mtTFB. Comparison of the three crystal structures and further study of the KsgA protein will provide insight into this interesting group of enzymes.
    Journal of Molecular Biology 06/2004; 339(2):337-53. · 3.91 Impact Factor
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    ABSTRACT: The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosylmethionine (SAM) to two adjacent adenosines in 16S rRNA. These modified adenosines are universally conserved in all species of eubacteria, eukaryotes and archaebacteria studied. Recombinant KsgA from Escherichia coli was overexpressed as a His-tagged fusion protein and purified. The recombinant protein was crystallized using PEG 4000 as a precipitant. The crystals belong to space group C2 and diffract X-rays to a resolution of 1.9 A. The unit-cell parameters are a = 173.9, b = 38.4, c = 83.0 A, beta = 90.0 degrees. Structure determination using the molecular-replacement method is at the early stages of refinement.
    Acta Crystallographica Section D Biological Crystallography 09/2003; 59(Pt 8):1490-2. · 14.10 Impact Factor