Interactions between DNA helicases and frozen topoisomerase IV-quinolone-DNA ternary complexes.

Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion.     

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.     

Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.     

Small-angle neutron scattering study of the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and valyl-tRNAVal

The formation of the ternary complex between bacterial elongation factor Tu, GTP, and valyl-tRNAVal has been studied by small-angle neutron scattering. Titrations of the protein with amino-acyl-tRNA solutions in both H2O and 70% D2O confirm the expected stoichiometry. The molecular weight obtained for the protein alone is significantly higher than expected and can be explained by postulating a monomer-dimer equilibrium. The titration data are then internally consistent with a dissociation of the dimer on ternary complex formation. The radius of gyration for the ternary complex and the calculation of the separation of the centers of mass of the protein and tRNA components suggest a compact model for the ternary complex.     

An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells

Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.     

The Human Adenovirus E4-ORF1 Protein Subverts Discs Large 1 to Mediate Membrane Recruitment and Dysregulation of Phosphatidylinositol 3-Kinase

Adenoviruses infect epithelial cells lining mucous membranes to cause acute diseases in people. They are also utilized as vectors for vaccination and for gene and cancer therapy, as well as tools to discover mechanisms of cancer due to their tumorigenic potential in experimental animals. The adenovirus E4-ORF1 gene encodes an oncoprotein that promotes viral replication, cell survival, and transformation by activating phosphatidylinositol 3-kinase (PI3K). While the mechanism of activation is not understood, this function depends on a complex formed between E4-ORF1 and the membrane-associated cellular PDZ protein Discs Large 1 (Dlg1), a common viral target having both tumor suppressor and oncogenic functions. Here, we report that in human epithelial cells, E4-ORF1 interacts with the regulatory and catalytic subunits of PI3K and elevates their levels. Like PI3K activation, PI3K protein elevation by E4-ORF1 requires Dlg1. We further show that Dlg1, E4-ORF1, and PI3K form a ternary complex at the plasma membrane. At this site, Dlg1 also co-localizes with the activated PI3K effector protein Akt, indicating that the ternary complex mediates PI3K signaling. Signifying the functional importance of the ternary complex, the capacity of E4-ORF1 to induce soft agar growth and focus formation in cells is ablated either by a mutation that prevents E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results demonstrate that E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1 hijacks the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for constitutive activation. This novel mechanism of Dlg1 subversion by adenovirus to dysregulate PI3K could be used by other pathogenic viruses, such as human papillomavirus, human T-cell leukemia virus type 1, and influenza A virus, which also target Dlg1 and activate PI3K in cells.     

Structural information for explaining the molecular mechanism of protein biosynthesis.

Protein biosynthesis is controlled by a number of proteins external to the ribosome. Of these, extensive structural investigations have been performed on elongation factor-Tu and elongation factor-G. This now gives a rather complete structural picture of the functional cycle of elongation factor-Tu and especially of the elongation phase of protein biosynthesis. The discovery that three domains of elongation factor-G are structurally mimicking the amino-acylated tRNA in the ternary complex of elongation factor-Tu has been the basis of much discussion of the functional similarities and functional differences of elongation factor-Tu and elongation factor-G in their interactions with the ribosome. Elongation factor-G:GDP is now thought to leave the ribosome in a state ready for checking the codon-anticodon interaction of the aminoacyl-tRNA contained in the ternary complex of elongation factor-Tu. Elongation factor-G does this by mimicking the shape of the ternary complex. Other translation factors such as the initiation factor-2 and the release factor 1 or 2 are also thought to mimic tRNA. These observations raise questions concerning the possible evolution of G-proteins involved in protein biosynthesis.