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.
The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.
The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negativepolarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.The influenza A viruses are members of the family Orthomyxoviridae whose genome comprises eight single-stranded RNA segments of negative polarity. The extreme genetic and antigenic diversity and the segmented nature of their genome are the basis of the yearly epidemics and occasional pandemics of influenza respiratory disease. The influenza A viruses are at evolutionary equilibrium in their natural reservoir, which includes several wild avian species, and from this reservoir, viruses or viral genes can occasionally be transferred to new influenza virus strains and result in the infection of mammals, including humans (22, 68). Although fears have been generated regarding the generation of a new pandemic by highly pathogenic H5N1 viruses, which have caused sporadic cases in humans (50), a new H1N1 virus of swine origin produced worldwide outbreaks (41) and led the WHO to declare a pandemic situation (http://www.who.int/csr/disease/swineflu/4th _meeting_ihr/en/index.html).The replication and transcription of the influenza A virus genome take place in the nucleus of infected cells and are carried out by macromolecular complexes called ribonucleoproteins (RNPs) that include each one of the virus RNA segments associated with monomers of the nucleoprotein (NP) and with the RNA-dependent RNA polymerase (reviewed in reference 12). This virus enzyme is formed by three subunits (PB1, PB2, and PA) and is responsible for both transcription and replication, as mutations in any of its subunits can lead to alterations in either process (3,13,14,16,17,33,51). For transcription, the virus polymerase recognizes cellular cap-containing newly synthesized cellular RNA polymerase II (Pol II) transcripts in a template-dependent manner (6, 31) and generates capped oligonucleotides that serve as primers to copy the RNP template (52, 54). The virus mRNAs are polyadenylated by the ...
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