scholarly journals Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 659-668 ◽  
Author(s):  
L S Folley ◽  
T D Fox
Keyword(s):  
Translation Initiation ◽  
Structural Features ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Initiation Codon ◽  
Translation Initiation Site ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Translation Initiation Codon

Abstract We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.

In vivo evaluation of the context sequence of the translation initiation codon in plants

Plant Science ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Marcin Lukaszewicz ◽  
Marc Feuermann ◽  
Bénédicte Jérouville ◽  
Arnaud Stas ◽  
Marc Boutry
Keyword(s):  
Translation Initiation ◽  
Initiation Codon ◽  
In Vivo Evaluation ◽  
Translation Initiation Codon

scholarly journals In Vivo Analysis of Saccharomyces cerevisiae COX2 mRNA 5′-Untranslated Leader Functions in Mitochondrial Translation Initiation and Translational Activation

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Heather M Dunstan ◽  
Noelle S Green-Willms ◽  
Thomas D Fox
Keyword(s):  
Translation Initiation ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Mitochondrial Translation ◽  
Context Analysis ◽  
Stem Loop ◽  
Sequence Elements ◽  
Translational Activator

Abstract We have used mutational and revertant analysis to study the elements of the 54-nucleotide COX2 5′-untranslated leader involved in translation initiation in yeast mitochondria and in activation by the COX2 translational activator, Pet111p. We generated a collection of mutants with substitutions spanning the entire COX2 5′-UTL by in vitro mutagenesis followed by mitochondrial transformation and gene replacement. The phenotypes of these mutants delimit a 31-nucleotide segment, from −16 to −46, that contains several short sequence elements necessary for COX2 5′-UTL function in translation. The sequences from −16 to −47 were shown to be partially sufficient to promote translation in a foreign context. Analysis of revertants of both the series of linker-scanning alleles and two short deletion/insertion alleles has refined the positions of several possible functional elements of the COX2 5′-untranslated leader, including a putative RNA stem-loop structure that functionally interacts with Pet111p and an octanucleotide sequence present in all S. cerevisiae mitochondrial mRNA 5′-UTLs that is a potential rRNA binding site.

scholarly journals Translation of the chloroplast psbC mRNA is controlled by interactions between its 5' leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii.

1997 ◽  
Vol 17 (6) ◽  
pp. 3440-3448 ◽  
Author(s):  
W Zerges ◽  
J Girard-Bascou ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Translation Initiation ◽  
Translational Control ◽  
Reporter Genes ◽  
Activation Function ◽  
Wild Type ◽  
Cis Acting ◽  
Translation Initiation Codon ◽  
Nuclear Loci

Translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5' untranslated region (5' UTR) and the functions encoded by two nuclear loci, which we name here TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC 5' UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1 function. These data demonstrate that sequences located in the middle of the psbC 5' UTR, TBC1, and TBC3 functionally interact to control the translation of the psbC mRNA.

scholarly journals The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditiselegans

Genetics ◽  
2010 ◽  
Vol 186 (4) ◽  
pp. 1187-1196 ◽  
Author(s):  
Lisa L. Maduzia ◽  
Anais Moreau ◽  
Nausicaa Poullet ◽  
Sebastien Chaffre ◽  
Yinhua Zhang
Keyword(s):  
Translation Initiation ◽  
Initiation Codon ◽  
Translation Initiation Codon ◽  
Codon Selection

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding

1993 ◽  
Vol 13 (7) ◽  
pp. 4087-4097
Author(s):  
J Wang ◽  
N Suzuki ◽  
Y Nishida ◽  
T Kataoka
Keyword(s):  
Heat Shock ◽  
Adenylyl Cyclase ◽  
Gene Replacement ◽  
Yeast Cells ◽  
Cyclase Activity ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Adenylyl Cyclase Activity

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

Novel Mutation of the Translation Initiation Codon of the α1-Globin Gene (ATG>AAG orHBA1:c.2T>A)

Hemoglobin ◽  
2016 ◽  
Vol 40 (5) ◽  
pp. 369-370 ◽  
Author(s):  
John S. Waye ◽  
Barry Eng ◽  
Meredith Hanna ◽  
Betty-Ann Hohenadel ◽  
Lisa Nakamura ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Novel Mutation ◽  
Globin Gene ◽  
Initiation Codon ◽  
Translation Initiation Codon

scholarly journals Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2

2020 ◽  
Vol 21 (3) ◽  
pp. 735
Author(s):  
Jerneja Tomsic ◽  
Arianna Smorlesi ◽  
Enrico Caserta ◽  
Anna Maria Giuliodori ◽  
Cynthia L. Pon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Geobacillus Stearothermophilus ◽  
Wild Type ◽  
Initiation Complex ◽  
Structural Alterations

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 ≥ to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

scholarly journals Fidelity of translation initiation is required for coordinated respiratory complex assembly

2019 ◽  
Vol 5 (12) ◽  
pp. eaay2118 ◽  
Author(s):  
Danielle L. Rudler ◽  
Laetitia A. Hughes ◽  
Kara L. Perks ◽  
Tara R. Richman ◽  
Irina Kuznetsova ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mitochondrial Protein ◽  
Molecular Machines ◽  
Ribosome Profiling ◽  
Untranslated Regions ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Initiation Factors ◽  
Mitochondrial Ribosomes

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle–specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

Sign in / Sign up

Export Citation Format

Share Document