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51.
The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations. 相似文献
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Estrada-Cuzcano A Neveling K Kohl S Banin E Rotenstreich Y Sharon D Falik-Zaccai TC Hipp S Roepman R Wissinger B Letteboer SJ Mans DA Blokland EA Kwint MP Gijsen SJ van Huet RA Collin RW Scheffer H Veltman JA Zrenner E;European Retinal Disease Consortium den Hollander AI Klevering BJ Cremers FP 《American journal of human genetics》2012,90(1):102-109
Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies. 相似文献
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Wen Li Xabier Agirrezabala Jianlin Lei Lamine Bouakaz Julie L Brunelle Rodrigo F Ortiz-Meoz Rachel Green Suparna Sanyal Mns Ehrenberg Joachim Frank 《The EMBO journal》2008,27(24):3322-3331
The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNAPhe, Trp-tRNATrp, or Leu-tRNALeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same ‘loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection. 相似文献
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This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second is that GDP/GTP exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a GTPase conformation with low nucleotide affinity. Since, however, recent biochemical and structural data seem to contradict this view, we present a generalized scheme for GTPase action. This novel ansatz accounts for those important cases when conformational switching in addition to guanine nucleotide exchange requires the presence of cofactors, and gives a more nuanced picture of how the nucleotide exchange is regulated. The scheme is also used to discuss some problems of interpretation that may arise when guanine nucleotide exchange mechanisms are inferred from experiments with analogs of GTP, like GDPNP, GDPCP, and GDP γ S. 相似文献
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With an in vitro poly(Phe) synthesis system we have tested recent models concerning translational accuracy in the stringent response during aminoacid starvation. We have found that cognate, deacylated tRNA of very high concentrations is unable to block the A-site. No influence of EF-Tu.ppGpp on ribosomal proofreading has been found. Alternative mechanisms to keep translational errors low by the stringent response are discussed. 相似文献
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