Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons

1984 ◽  
Vol 4 (7) ◽  
pp. 1191-1197
Author(s):  
R S Zitomer ◽  
D A Walthall ◽  
B C Rymond ◽  
C P Hollenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fold Increase ◽  
Transcription Unit ◽  
Initiation Codon ◽  
Wild Type ◽  
Protein Coding ◽  
Translational Initiation ◽  
Nonsense Codon ◽  
Initiation Of Translation

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.

scholarly journals Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.

1984 ◽  
Vol 4 (7) ◽  
pp. 1191-1197 ◽  
Author(s):  
R S Zitomer ◽  
D A Walthall ◽  
B C Rymond ◽  
C P Hollenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fold Increase ◽  
Transcription Unit ◽  
Initiation Codon ◽  
Wild Type ◽  
Protein Coding ◽  
Translational Initiation ◽  
Nonsense Codon ◽  
Initiation Of Translation

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

scholarly journals Engineering Desiccation Tolerance inEscherichia coli

2000 ◽  
Vol 66 (4) ◽  
pp. 1680-1684 ◽  
Author(s):  
Daniela Billi ◽  
Deborah J. Wright ◽  
Richard F. Helm ◽  
Todd Prickett ◽  
Malcolm Potts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phase Transition ◽  
Freeze Drying ◽  
Fold Increase ◽  
Inescherichia Coli ◽  
Vibration Frequencies ◽  
Wild Type ◽  
Air Drying ◽  
Phase Transition Temperatures ◽  
Cell Dehydration

ABSTRACT Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized inEscherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

2008 ◽  
Vol 414 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Oliver Mueller-Cajar ◽  
Spencer M. Whitney
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Fold Increase ◽  
Functional Expression ◽  
Kinetic Properties ◽  
Selection System ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

scholarly journals Improving Escherichia coli FucO for Furfural Tolerance by Saturation Mutagenesis of Individual Amino Acid Positions

2013 ◽  
Vol 79 (10) ◽  
pp. 3202-3208 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Ryan D. Geddes ◽  
Keelnatham T. Shanmugam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Gene ◽  
Free Energy Calculations ◽  
Saturation Mutagenesis ◽  
Interfacial Region ◽  
Wild Type ◽  
Gene Encoding ◽  
Translational Initiation ◽  
Content Type ◽  
Furfural Tolerance

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose with mineral acids. InEscherichia coli, furfural tolerance can be increased by expressing the nativefucOgene (encoding lactaldehyde oxidoreductase). This enzyme also catalyzes the NADH-dependent reduction of furfural to the less toxic alcohol. Saturation mutagenesis was combined with growth-based selection to isolate a mutated form offucOthat confers increased furfural tolerance. The mutation responsible, L7F, is located within the interfacial region of FucO homodimers, replacing the most abundant codon for leucine with the most abundant codon for phenylalanine. Plasmid expression of the mutant gene increased FucO activity by more than 10-fold compared to the wild-typefucOgene and doubled the rate of furfural metabolism during fermentation. No inclusion bodies were evident with either the native or the mutated gene. mRNA abundance for the wild-type and mutantfucOgenes differed by less than 2-fold. TheKm(furfural) for the mutant enzyme was 3-fold lower than that for the native enzyme, increasing efficiency at low substrate concentrations. The L7F mutation is located near the FucO N terminus, within the ribosomal binding region associated with translational initiation. Free-energy calculations for mRNA folding in this region (nucleotides −7 to +37) were weak for the native gene (−4.1 kcal mol−1) but weaker still for thefucOmutant (−1.0 to −0.1 kcal mol−1). The beneficial L7F mutation in FucO is proposed to increase furfural tolerance by improving gene expression and increasing enzyme effectiveness at low substrate levels.

scholarly journals TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation.

1994 ◽  
Vol 14 (6) ◽  
pp. 3634-3645 ◽  
Author(s):  
M K Robinson ◽  
W H van Zyl ◽  
E M Phizicky ◽  
J R Broach
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Cell Separation ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Protein Phosphatase 2C ◽  
Trna Splicing ◽  
Multiple Phenotypes ◽  
Higher Eukaryotes

The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.

scholarly journals Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization

1995 ◽  
Vol 310 (2) ◽  
pp. 605-614 ◽  
Author(s):  
M J Stone ◽  
W Ruf ◽  
D J Miles ◽  
T S Edgington ◽  
P E Wright
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Tissue Factor ◽  
Inclusion Bodies ◽  
Factor Viia ◽  
Factor X ◽  
Wild Type ◽  
Proteolytic Activation ◽  
E Coli

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

scholarly journals Genetic Analysis of Rap1p/Sir3p Interactions in Telomeric and HML Silencing in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 81-93 ◽  
Author(s):  
Cheng Liu ◽  
Arthur J Lustig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Fold Increase ◽  
Missense Mutations ◽  
Tail Domain ◽  
Wild Type ◽  
Telomeric Silencing

Abstract We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telomeric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727–747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1s domain. rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telomeric complexes by Sir3p-Sir3p interactions.

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