scholarly journals cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 97-112 ◽  
Author(s):  
I Pinto ◽  
J G Na ◽  
F Sherman ◽  
M Hampsey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Point Mutations ◽  
Start Codon ◽  
Reading Frame ◽  
Codon Recognition ◽  
Short Open Reading Frame ◽  
Initiator Codon ◽  
Extragenic Suppressors ◽  
Cis And Trans

Abstract The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

mRNA structures influencing translation in the yeast Saccharomyces cerevisiae

1988 ◽  
Vol 8 (4) ◽  
pp. 1591-1601
Author(s):  
S B Baim ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Context Effect ◽  
Leader Region ◽  
Mrna Sequence ◽  
Specific Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Initiator Codon ◽  
The Stability ◽  
Nested Deletions

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.

scholarly journals Two isoforms of Saccharomyces cerevisiae glutaredoxin 2 are expressed in vivo and localize to different subcellular compartments

2002 ◽  
Vol 364 (3) ◽  
pp. 617-623 ◽  
Author(s):  
José R. PEDRAJAS ◽  
Pablo PORRAS ◽  
Emilia MARTÍNEZ-GALISTEO ◽  
C. Alicia PADILLA ◽  
Antonio MIRANDA-VIZUETE ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Exponential Phase ◽  
Start Codon ◽  
Immunochemical Analysis ◽  
Translation Product ◽  
Differential Processing ◽  
Reading Frame ◽  
Yeast Extract Peptone Dextrose ◽  
Long Form

Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a ‘classical’ Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.

scholarly journals mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (4) ◽  
pp. 1591-1601 ◽  
Author(s):  
S B Baim ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Context Effect ◽  
Leader Region ◽  
Mrna Sequence ◽  
Specific Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Initiator Codon ◽  
The Stability ◽  
Nested Deletions

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.

scholarly journals A DNA Helicase Required for Maintenance of the Functional Mitochondrial Genome in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (5) ◽  
pp. 1816-1824 ◽  
Author(s):  
Tiina Sedman ◽  
Silja Kuusk ◽  
Sirje Kivi ◽  
Juhan Sedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Genome ◽  
Signal Sequence ◽  
Carbon Sources ◽  
Point Mutations ◽  
Nuclear Genome ◽  
Dna Helicase ◽  
Dependent Manner ◽  
Reading Frame

ABSTRACT A novel DNA helicase, a homolog of several prokaryotic helicases, including Escherichia coli Rep and UvrD proteins, is encoded by the Saccharomyces cerevisiae nuclear genome open reading frame YOL095c on the chromosome XV. Our data demonstrate that the helicase is localized in the yeast mitochondria and is loosely associated with the mitochondrial inner membrane during biochemical fractionation. The sequence of the C-terminal end of the 80-kDa helicase protein is similar to a typical N-terminal mitochondrial targeting signal; deletions and point mutations in this region abolish transport of the protein into mitochondria. The C-terminal signal sequence of the helicase targets a heterologous carrier protein into mitochondria in vivo. The purified recombinant protein can unwind duplex DNA molecules in an ATP-dependent manner. The helicase is required for the maintenance of the functional ([rho +]) mitochondrial genome on both fermentable and nonfermentable carbon sources. However, the helicase is not essential for the maintenance of several defective ([rho −]) mitochondrial genomes. We also demonstrate that the helicase is not required for transcription in mitochondria.

scholarly journals Point Mutations Identify a Conserved Region of the Saccharomyces cerevisiae AFR1 Gene That Is Essential for Both the Pheromone Signaling and Morphogenesis Functions

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 43-55
Author(s):  
Cordell R DeMattei ◽  
Colleen P Davis ◽  
James B Konopka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Distinct Point ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Pheromone Signaling ◽  
Essential Region ◽  
The Relationship ◽  
Mutant Genes

Abstract Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling. The AFR1 mutants were also defective when expressed as fusions to STE2, the α-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S. uvarum and S. douglasii. In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region. Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both functions may be due to the same action of Afr1p. Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.

Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Parnika Kadam ◽  
Susette Mueller ◽  
Hong Ji ◽  
Kathryn Sandberg
Keyword(s):  
Laser Scanning ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Receptor Internalization ◽  
Start Codon ◽  
Ang Ii ◽  
Exon 2 ◽  
Reading Frame ◽  
Short Open Reading Frame ◽  
Angiotensin Type

Background: We recently found that a seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5’ leader sequence (5'LS) of the angiotensin type 1a receptor (AT1aR) mRNA inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid. Methods: Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software. Results: Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001]. Conclusion: The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.

scholarly journals A MUTATOR AFFECTING THE REGION OF THE ISO-1-CYTOCHROME c GENE IN YEAST

Genetics ◽  
1979 ◽  
Vol 92 (3) ◽  
pp. 783-802 ◽  
Author(s):  
Susan W Liebman ◽  
Arjun Singh ◽  
Fred Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Chromosomal Aberrations ◽  
High Rate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Uv Sensitivity ◽  
Mutator Gene ◽  
Mendelian Gene ◽  
Cis And Trans ◽  
Low Rate

ABSTRACT The mutator gene DEL1 in the yeast Saccharomyces cerevisiae cauces a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYC1, which determines the structure of iso-l-cyto-chrome c; RAD7, which controls UV sensitivity; and OSM1, which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments.——The multisite mutations arise at a rate of approximately 10-5 to 10-6 per cell per division in DEL1 strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cycl mutants at a low rate of approximately 10-8 to 10-9. No evidence for multisite mutations was obtained upon analysis of numerous spontaneous adel, ade2, met2 and met15 mutants isolated in a DEL1 strain. DEL1 segregates as a single Mendelian gene closely linked to the CYC1 locus. DEL1 appears to be both cis- and trans-dominant. The location of the DEL1 gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself.

scholarly journals Genetic characterization of the Saccharomyces cerevisiae translational initiation suppressors sui1, sui2 and SUI3 and their effects on HIS4 expression.

Genetics ◽  
1990 ◽  
Vol 124 (3) ◽  
pp. 483-495 ◽  
Author(s):  
B Castilho-Valavicius ◽  
H Yoon ◽  
T F Donahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Genetic Characterization ◽  
Point Mutations ◽  
Base Change ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Translational Initiation ◽  
Initiator Codon

Abstract Saccharomyces cerevisiae strains containing mutations of the HIS4 translation initiation AUG codon were studied by reversion analysis in an attempt to identify components of the translation initiation complex that might participate in initiation site selection during the scanning process. The genetic characterization of these revertants identified three unlinked suppressor loci: SUI1, SUI2 and sui3, which when mutated restored the expression of the HIS4 allele despite the absence of the AUG initiator codon. Both sui1 and sui2 are recessive and cause temperature-sensitive growth on enriched medium. The temperature-sensitive phenotype and the ability to restore HIS4 expression associated with either sui1 or sui2 mutations cosegregate in crosses. SUI3 mutations are dominant and do not alter the thermal profile for growth. None of the mutations at the three loci suppresses known frameshift, missense or nonsense mutations. Each is capable of suppressing the nine different point mutations of the initiator codon at HIS4 or HIS4-lacZ as well as a two base change (ACC) and a three base deletion of the AUG codon, suggesting that the site of suppression resides outside the normal initiator region. sui1 and sui2 suppressor mutations were mapped to chromosomes XIV and X, respectively. Suppression by sui1, sui2 and SUI3 mutations results in 14-, 11- and 47-fold increases, respectively, relative to isogenic parent strains, in the expression of a HIS4 allele lacking the initiator AUG codon. Part of this increase in the HIS4 expression by sui2 and SUI3 can be attributed to increases of HIS4 mRNA levels, presumably mediated by perturbation of the general amino acid control system of yeast.

Sign in / Sign up

Export Citation Format

Share Document