Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...
Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.
Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.modeling | molecular dynamics | flexible fitting I n all living cells, the translation of mRNA into polypeptide occurs on ribosomes. Ribosomes provide a platform upon which aminoacyl-tRNAs interact with the mRNA as well as position the aminoacyl-tRNAs for peptide-bond formation (1). Ribosomes are composed of two subunits, a small subunit that monitors the mRNA-tRNA codon-anticodon duplex to ensure fidelity of decoding (2, 3) and a large subunit that contains the active site where peptide-bond formation occurs (4). Both the small and large subunits are composed of RNA and protein: In eubacteria such as Escherichia coli, the small subunit contains one 16S rRNA and 21 ribosomal proteins (r proteins), whereas the large subunit contains 5S and 23S rRNAs and 33 r proteins. Crystal structures of the complete bacterial 70S ribosome were initially reported at 5.5 Å (5), with an interpretation based on atomic models of the individual subunit structures (6-8), and are now available at atomic resolution (9). These structures have provided unparalleled insight into the mechanism of different steps of translation (1) as well as inhibition by antibiotics (10).Compared to the bacterial ribosome, the eukaryotic counterpart is more complicated, containing expansion segments (ES) and variable regions in the rRNA as well as many additional r proteins and r-protein extensions. Plant and fungal 80S ribosomes contain ∼5;500 nucleotides (nts) of rRNA and ∼80 r proteins, whereas bacterial 70S ribosomes comprise ∼4;500 nts and 54 r proteins. The additional elements present in eukaryotic ribosomes may reflect the increased complexity of translation regulation in eukaryotic cells, as evident for assembly, translation initiation, and development, as well as the phenomenon of localized translation (11-15).Early models for eukaryotic ribosomes were derived from electron micrographs of negative-stain or freeze-dried ribosomal particles (16) and localization of r proteins was attempted using immuno-EM and cross-linking approaches; see, for example, refs. 17-20. The first cryo-EM reconstruction of a eukaryotic 80S ribosome was reported for wheat germ (Triticum aestivum) at 38 Å (21). Initial core models for the yeast 80S ribosome were built at 15-Å resolution (22) by docking the rRNA s...
The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, that are members of the Lrp/AsnC family of bacterial transcription regulators. In contrast, this archaeon's RNA polymerase and core transcription factors are of eukaryotic type. Using the M. jannaschii high-temperature in vitro transcription system, we show that Ptr2 is a potent transcriptional activator, and that it conveys its stimulatory effects on its cognate eukaryal-type transcription machinery from an upstream activating region composed of two Ptr2-binding sites. Transcriptional activation is generated, at least in part, by Ptr2-mediated recruitment of the TATA-binding protein to the promoter.T he core components of archaeal transcription closely resemble those of eukaryotic RNA polymerase II (1). Archaeal promoters consist of an AϩT-rich TATA-like element recognized by archaeal TATA-binding protein (TBP); the TFIIBrelated transcription factor B (TFB) binds to the TBP-DNA complex and directs a eukaryotic-type RNA polymerase (RNAP) to specifically initiate transcription at an initiator sequence located some 25 bp downstream of the TATA element. Efficient preinitiation complex (PIC) assembly is ensured by the adjacent purine-rich BRE element, which mediates sequencespecific interactions with TFB upstream of the TATA box (2) and dictates the directionality of transcription complex assembly and initiation (3). TBP, TFB, and RNAP are necessary and sufficient to direct transcription at many archaeal promoters in vitro; a modest stimulatory effect of TFE, the archaeal homologue of the ␣ subunit of the RNA polymerase II transcription factor TFIIE, is discerned under conditions of suboptimal TBP-TATA box interaction (4, 5).On the other hand, all archaeal genomes sequenced to date encode potential transcription regulators of bacterial type, underscoring the chimeric nature of the archaeal transcription apparatus (6, 7). Particular interest is attached to the question of how these bacterial-type effectors, especially activators, generate regulation of a eukaryote-like transcription system. All of the putative regulators of transcription that have been characterized in vitro, the metal-dependent repressor 1 (MDR1) from Archaeoglobus fulgidus (8), as well as the homologues LrpA from Pyrococcus furiosus (9, 10), Lrs-14 from Sulfolobus solfataricus (11), and Ptr1 from Methanococcus jannaschii (unpublished results), have only been shown to repress transcription by their cognate RNA polymerases.Here we show that Ptr2, a site-specific helix-turn-helix DNAbinding protein from the hyperthermophilic archaeon M. jannaschii and homologue of the bacterial leucine-responsive regulatory protein (Lrp) family of transcription factors, is a potent transcriptional activator in vitro. We also show that Ptr2 conveys its stimulatory effects on its cognate transcription machinery through direct recruitment of TBP. Materials and MethodsProtein Purification. The RNA polymerase from Methanococcus͞ Methanocaldococcus jannaschi...
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