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ABSTRACT: The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI:http://dx.doi.org/10.7554/eLife.00482.001.
eLife. 01/2013; 2:e00482.
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ABSTRACT: The initiation of protein biosynthesis entails the ordered assembly of elongation-competent ribosomes, with an initiator tRNA basepaired to an appropriate mRNA start codon. In eukaryotes, this process is more complex than in prokaryotes and involves numerous protein factors that mediate tRNA delivery, mRNA binding, start codon selection and subunit joining. The recent 40S:eIF1, 80S and eIF2:tRNA:GDPNP ternary complex structures provide an initial structural framework toward a molecular understanding of the eukaryotic translation initiation process. Updated homology models of larger initiation complexes provide first insights into the likely arrangements of these higher-order complexes, but also reveal the limits of our current understanding of the eukaryotic translation initiation process.
Current Opinion in Structural Biology 08/2012; · 9.42 Impact Factor
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ABSTRACT: Eukaryotic ribosomes are significantly larger and more complex than their prokaryotic counterparts. This parallels the increased complexity of the associated cellular machinery responsible for translation initiation, ribosome assembly, and the regulation of protein synthesis in eukaryotic cells. The recently determined crystal structures of the small (40S) and large (60S) ribosomal subunits and the 80S ribosome now provide an atomic description of this essential molecular machine and reveal its eukaryote-specific features. In this review, we discuss the common structural principles underlying the evolution of both ribosomal subunits. The recently obtained structural information provides a framework for further genetic, biochemical and structural studies of eukaryotic ribosomes. At the same time, it facilitates a direct comparison between prokaryotic and eukaryotic ribosomal features.
Trends in Biochemical Sciences 03/2012; 37(5):189-98. · 10.85 Impact Factor
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ABSTRACT: Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.
Science 11/2011; 334(6058):941-8. · 31.20 Impact Factor
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Rafael Núñez-Ramírez, Sebastian Klinge,
Ludovic Sauguet,
Roberto Melero,
María A Recuero-Checa,
Mairi Kilkenny,
Rajika L Perera,
Begoña García-Alvarez,
Richard J Hall,
Eva Nogales,
Luca Pellegrini,
Oscar Llorca
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ABSTRACT: The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.
Nucleic Acids Research 06/2011; 39(18):8187-99. · 8.03 Impact Factor
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ABSTRACT: DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood.
Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase.
Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.
PLoS ONE 01/2010; 5(4):e10083. · 4.09 Impact Factor
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ABSTRACT: Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol alpha. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol alpha reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B-CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.
The EMBO Journal 07/2009; 28(13):1978-87. · 9.20 Impact Factor
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ABSTRACT: Primases synthesize the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.
Nature Structural & Molecular Biology 10/2007; 14(9):875-7. · 12.71 Impact Factor
