The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Warren P. Tate - One of the best experts on this subject based on the ideXlab platform.
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Comparison of characteristics and function of translation Termination Signals between and within prokaryotic and eukaryotic organisms
Nucleic Acids Research, 2006Co-Authors: Andrew G. Cridge, Louise L. Major, Elizabeth S Poole, Leif A. Isaksson, Alhad A. Mahagaonkar, Warren P. TateAbstract:Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis Termination Signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core Signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in Signal abundance and Termination efficiency. In Escherichia coli Signal abundance correlated with Termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination Signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.
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Evolutionary optimisation of MLP for modelling protein synthesis Termination Signal efficiency
Proceedings of the 2002 Congress on Evolutionary Computation. CEC'02 (Cat. No.02TH8600), 2002Co-Authors: Michael J. Watts, Louise L. Major, Warren P. TateAbstract:Multi-layer perceptron neural networks that have been optimised by an evolutionary algorithm, were used to model experimentally determined protein synthesis Termination Signal strength. It is shown that the evolutionary algorithm produced much smaller multi-layer perceptrons with comparable or superior performance to manually designed networks.
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eLS - Protein Synthesis Termination
Encyclopedia of Life Sciences, 2001Co-Authors: Warren P. Tate, Elizabeth S Poole, Sally A ManneringAbstract:Protein synthesis Termination is the process by which a completed polypeptide is released from the ribosome after the coding information within a messenger ribonucleic acid (mRNA) has been successfully translated. Keywords: Termination Signal; release factor; ribosome recycling factor; completed polypeptide; recoding
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Translational Termination in Escherichia coli: Three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing Signals
Nucleic Acids Research, 1998Co-Authors: Elizabeth S Poole, Louise L. Major, Sally A Mannering, Warren P. TateAbstract:The observations that the Escherichia coli release factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop Signals, stimulated us to determine whether there was a more extended Termination Signal for RF2 recognition. Analysis of the 34 contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element. Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10). Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2. A strong selection bias for particular bases in the +4 to +6 positions of certain E.coli UGANNN Termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo Termination Signal strength. These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence Termination Signal decoding efficiency.
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decoding the translational Termination Signal the polypeptide chain release factor in escherichia coli crosslinks to the base following the stop codon
RNA, 1997Co-Authors: Elizabeth S Poole, R Brimacombe, Warren P. TateAbstract:Protein release factors act like tRNA analogues in decoding translational stop Signals. Statistical analysis of the sequences at translational stop sites and functional studies with particular Signals indicate this mimicry involves an increase in the length of the Signal in the mRNA. The base following the stop codon (+4 base) is of particular interest because it has a strong influence on the competitiveness of the stop Signal at recoding sites, suggesting it might form part of the release factor recognition element. Site-directed crosslinking from the +4 base showed that it is in close proximity to the Escherichia coli release factor-2 in a Termination complex, a prerequisite for the +4 base being part of the recognition element. Fingerprinting analysis indicates that crosslinking to the release factor occurred from both +1 and +4 positions of the stop Signal in the same RNA molecule. This provides more evidence that the +4 base may be an integral part of the decoding signature in the mRNA during the Termination phase of protein biosynthesis.
William T Mcallister - One of the best experts on this subject based on the ideXlab platform.
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characterization of an unusual sequence specific Termination Signal for t7 rna polymerase
Journal of Biological Chemistry, 1998Co-Authors: Biao He, Alexander Kukarin, Dmitry Temiakov, Stephen T Chinbow, D L Lyakhov, Minqing Rong, Russell K Durbin, William T McallisterAbstract:Abstract We have characterized an unusual type of Termination Signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish Termination. The primary site of Termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of Termination, and in particular the nucleotide at the site of Termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the Signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for Termination at this class of Signal.
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characterization of two types of Termination Signal for bacteriophage t7 rna polymerase
Journal of Molecular Biology, 1994Co-Authors: Lynn E Macdonald, Russell K Durbin, John J Dunn, William T McallisterAbstract:Abstract The late bacteriophage T7 terminator (T7-TΦ) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; Termination occurs at a 3′ G residue just downstream of the U run. In this work, we have explored the features of this Signal that are required for efficient Termination by T7 RNA polymerase. Whereas replacement of the template-encoded 3′ G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented Termination. Deletion analysis indicates that the stem-loop and U-tract are not sufficient for Termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of Termination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA polymerase. We have mapped the primary site of Termination in the PTH Signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region required for PTH terminator function extends only 23 bp upstream from the Termination site, and subcloning of a 31 bp fragment that includes this region of the PTH Signal provides efficient Termination. A modified form of T7 RNA polymerase resulting from a single proteolytic cleavage between residues 178 and 179, or mutant polymerases that are altered in this region of the enzyme, fail to recognize the PTH Signal while still terminating at T7-TΦ.
Gerald Striedner - One of the best experts on this subject based on the ideXlab platform.
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preventing t7 rna polymerase read through transcription a synthetic Termination Signal capable of improving bioprocess stability
ACS Synthetic Biology, 2015Co-Authors: Juergen Mairhofer, Alexander Wittwer, Monika Cserjanpuschmann, Gerald StriednerAbstract:The phage-derived T7 RNA polymerase is the most prominent orthogonal transcriptions system used in the field of synthetic biology. However, gene expression driven by T7 RNA polymerase is prone to read-through transcription due to contextuality of the T7 terminator. The native T7 terminator has a Termination efficiency of approximately 80% and therefore provides insufficient insulation of the expression unit. By using a combination of a synthetic T7 Termination Signal with two well-known transcriptional terminators (rrnBT1 and T7), we have been able to increase the Termination efficiency to 99%. To characterize putative effects of an enhanced Termination Signal on product yield and process stability, industrial-relevant fed batch cultivations have been performed. Fermentation of a E. coli HMS174(DE3) strain carrying a pET30a derivative containing the improved Termination Signal showed a significant decrease of plasmid copy number (PCN) and an increase in total protein yield under standard conditions.
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Preventing T7 RNA Polymerase Read-through Transcription—A Synthetic Termination Signal Capable of Improving Bioprocess Stability
ACS Synthetic Biology, 2014Co-Authors: Juergen Mairhofer, Alexander Wittwer, Monika Cserjan-puschmann, Gerald StriednerAbstract:The phage-derived T7 RNA polymerase is the most prominent orthogonal transcriptions system used in the field of synthetic biology. However, gene expression driven by T7 RNA polymerase is prone to read-through transcription due to contextuality of the T7 terminator. The native T7 terminator has a Termination efficiency of approximately 80% and therefore provides insufficient insulation of the expression unit. By using a combination of a synthetic T7 Termination Signal with two well-known transcriptional terminators (rrnBT1 and T7), we have been able to increase the Termination efficiency to 99%. To characterize putative effects of an enhanced Termination Signal on product yield and process stability, industrial-relevant fed batch cultivations have been performed. Fermentation of a E. coli HMS174(DE3) strain carrying a pET30a derivative containing the improved Termination Signal showed a significant decrease of plasmid copy number (PCN) and an increase in total protein yield under standard conditions.
Toshiaki Katada - One of the best experts on this subject based on the ideXlab platform.
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the eukaryotic polypeptide chain releasing factor erf3 gspt carrying the translation Termination Signal to the 3 poly a tail of mrna direct association of erf3 gspt with polyadenylate binding protein
Journal of Biological Chemistry, 1999Co-Authors: Shinichi Hoshino, Mariko Imai, Tetsuo Kobayashi, Naoyuki Uchida, Toshiaki KatadaAbstract:Abstract The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation Termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the Termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation Termination Signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs.
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The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation Termination Signal to the 3'-poly(A) tail of mRNA-Direct association of eRF3/GSPT with polyadenylate-binding protein
Journal of Biological Chemistry, 1999Co-Authors: Shinichi Hoshino, Mariko Imai, Tetsuo Kobayashi, Naoyuki Uchida, Toshiaki KatadaAbstract:Abstract The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1α, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation Termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the Termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation Termination Signal to 3′-poly(A) tail ubiquitously present in eukaryotic mRNAs.
Russell K Durbin - One of the best experts on this subject based on the ideXlab platform.
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characterization of an unusual sequence specific Termination Signal for t7 rna polymerase
Journal of Biological Chemistry, 1998Co-Authors: Biao He, Alexander Kukarin, Dmitry Temiakov, Stephen T Chinbow, D L Lyakhov, Minqing Rong, Russell K Durbin, William T McallisterAbstract:Abstract We have characterized an unusual type of Termination Signal for T7 RNA polymerase that requires a conserved 7-base pair sequence in the DNA (ATCTGTT in the non-template strand). Each of the nucleotides within this sequence is critical for function, as any substitutions abolish Termination. The primary site of Termination occurs 7 nucleotides downstream from this sequence but is context-independent (that is, the sequence around the site of Termination, and in particular the nucleotide at the site of Termination, need not be conserved). Termination requires the presence of the conserved sequence and its complement in duplex DNA and is abolished or diminished if the Signal is placed downstream of regions in which the non-template strand is missing or mismatched. Under the latter conditions, much of the RNA product remains associated with the template. The latter results suggest that proper resolution of the transcription bubble at its trailing edge and/or displacement of the RNA product are required for Termination at this class of Signal.
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characterization of two types of Termination Signal for bacteriophage t7 rna polymerase
Journal of Molecular Biology, 1994Co-Authors: Lynn E Macdonald, Russell K Durbin, John J Dunn, William T McallisterAbstract:Abstract The late bacteriophage T7 terminator (T7-TΦ) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; Termination occurs at a 3′ G residue just downstream of the U run. In this work, we have explored the features of this Signal that are required for efficient Termination by T7 RNA polymerase. Whereas replacement of the template-encoded 3′ G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented Termination. Deletion analysis indicates that the stem-loop and U-tract are not sufficient for Termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of Termination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA polymerase. We have mapped the primary site of Termination in the PTH Signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region required for PTH terminator function extends only 23 bp upstream from the Termination site, and subcloning of a 31 bp fragment that includes this region of the PTH Signal provides efficient Termination. A modified form of T7 RNA polymerase resulting from a single proteolytic cleavage between residues 178 and 179, or mutant polymerases that are altered in this region of the enzyme, fail to recognize the PTH Signal while still terminating at T7-TΦ.