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Initiation Complex (initiation + complex)
Selected AbstractsInitiation of JC virus DNA replication in vitro by human and mouse DNA polymerase ,-primaseFEBS JOURNAL, Issue 9 2003Richard W. P. Smith Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase ,-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase ,-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex. [source] Central role for Cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extractsGENES TO CELLS, Issue 6 2000Satoru Mimura Background In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. Results Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase , and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase , onto chromatin required Cdc45 but did not require DNA polymerase ,. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase , on chromatin. Conclusions These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA. [source] Quantitative resistance to Plum pox virus in Prunus davidiana P1908 linked to components of the eukaryotic translation initiation complexPLANT PATHOLOGY, Issue 3 2009G. Marandel A complex, polygenic resistance to Plum pox virus (PPV) was previously described in a wild peach-related species, Prunus davidiana clone P1908. In the current study, an analysis of quantitative trait loci (QTL) was performed on an F2 population comprising 99 individuals obtained by selfing the F1 individual #40 of an interspecific cross between susceptible nectarine cv. Summergrand and the resistant P. davidiana clone P1908. Six QTL were identified using both parametric and non-parametric methods of detection, individually explaining 5,28% of the phenotypic variance. The total phenotypic variation explained ranged from 29 to 58%. Alignment of the genetic map of the F2 cross with the P. davidiana parent map showed consistency of QTL over generations, with three of the six QTL co-localizing at the 1-LOD interval and another one at the 2-LOD interval. Two of the QTL were mapped onto linkage group one, where resistance to PPV was previously mapped in apricot. Development and mapping of new microsatellite markers linked to candidate genes revealed a striking co-localization of three of the detected QTL with gene copies coding for eukaryotic translation initiation factors eIF4E and eIF(iso)4G. As co-localization of one QTL with candidate gene eIF(iso)4E was previously reported in the F1 population, the results reported here strongly reinforce the idea that components of the eukaryotic translation initiation complex are correlated with resistance to PPV in P. davidiana P1908. [source] Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcriptionPROTEIN SCIENCE, Issue 9 2000Hung-Ta Chen Abstract Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/ TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2,59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel ,,strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the ,,sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the ,,sheet. [source] Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme actionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2009Bhaskar Chetnani The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerable plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel. [source] Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreBGENES TO CELLS, Issue 5 2001Ranjan Sen Background The initiation arrest at a modified ,PR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown. Results The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the ,PR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6,9-fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not. Conclusions All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest. [source] Cyclin-dependent kinase 1 plays a critical role in DNA replication control during rat liver regeneration,HEPATOLOGY, Issue 6 2009Delphine Garnier Liver regeneration is a unique process to restore hepatic homeostasis through rapid and synchronous proliferation of differentiated hepatocytes. Previous studies have shown that hepatocyte proliferation is characterized by high expression levels of the "mitotic" cyclin-dependent kinase 1 (Cdk1) during S-phase compared to other mammalian cells. In the light of findings showing that Cdk1 compensates for the loss of Cdk2 and drives S-phase in Cdk2-deficient cells derived from Cdk2 knockout mice, we took advantage of the models of liver regeneration following partial hepatectomy and primary cultures of normal rat hepatocytes to further examine the involvement of Cdk1 during DNA replication in hepatocytes and to dissect specific cell cycle regulation in hepatocytes compared to control human foreskin fibroblasts. In hepatocytes, Cdk1 exhibited a biphasic activation pattern correlating S-phase and G2/M transition, bound to cyclin A or B1 and localized to the nucleus during DNA replication. Importantly, small interfering RNA (siRNA)-mediated silencing of Cdk1 led to a strong decrease in DNA synthesis without affecting centrosome duplication. Furthermore, in hepatocytes arrested by the iron chelator O-Trensox in early S-phase prior to DNA replication, Cdk1/cyclin complexes were active, while replication initiation components such as the minichromosome maintenance 7 (Mcm7) protein were loaded onto DNA. Moreover, Mcm7 expression and loading onto DNA were not modified by Cdk1 silencing. Conversely, in fibroblasts, Cdk1 expression and activation were low in S-phase and its silencing did not reduce DNA synthesis. Conclusion: Cdk1 is essential for DNA replication downstream formation of replication initiation complexes in hepatocytes but not in fibroblasts and, as such, our data exemplify crucial differences in the cell cycle regulation between various mammalian cell types. (HEPATOLOGY 2009.) [source] Translational repression mechanisms in prokaryotesMOLECULAR MICROBIOLOGY, Issue 5 2003Paula Jean Schlax Summary Translational repression results from a complex choreography of macromolecular interactions interfering with the formation of translational initiation complexes. The relationship between the rate and extent of formation of these interactions to form repressed mRNA complexes determines the extent of repression. A novel analysis of repression mechanisms is presented here and it indicates that the reversibility of repressed complex formation influences the steady state balance of the distribution of translationally active and inactive complexes and therefore has an impact on the efficiency of repression. Reviewed here is evidence for three distinct translational repression mechanisms, regulating expression of the transcription factor ,32, threonine tRNA synthetase and ribosomal proteins on the , operon in Escherichia coli. Efficient regulation of expression in these systems makes use of specific mRNA structures in quite different ways. [source] A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTPBIOTECHNOLOGY PROGRESS, Issue 5 2009Hirohisa Koga Abstract Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 ,M of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B1-320) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||