scholarly journals Vps34 and TOR Kinases Coordinate HAC1 mRNA Translation in the presence or absence of Ire1-dependent Splicing

2021 ◽  
Author(s):  
Jagadeesh Kumar Uppala ◽  
Sankhajit Bhattacharjee ◽  
Madhusudan Dey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Responses ◽  
Mrna Translation ◽  
Nutrient Sensing ◽  
Rna Cleavage ◽  
Phosphatidylinositol 3 Kinase ◽  
Unfolded Proteins ◽  
Base Pairs ◽  
Rna Hairpins ◽  
Parallel Networks

In the budding yeast Saccharomyces cerevisiae an mRNA, called HAC1, exists in a translationally repressed form in the cytoplasm. Under conditions of cellular stress, such as when unfolded proteins accumulate inside the endoplasmic reticulum (ER), an RNase Ire1 removes an intervening sequence (intron) from the HAC1 mRNA by non-conventional cytosolic splicing. Removal of the intron results in translational de-repression of HAC1 mRNA and production of a transcription factor that activates expressions of many enzymes and chaperones to increase the protein-folding capacity of the cell. Here, we show that Ire1-mediated RNA cleavage requires Watson-Crick base pairs in two RNA hairpins, which are located at the HAC1 mRNA exon-intron junctions. Then, we show that the translational de-repression of HAC1 mRNA can occur independent of cytosolic splicing. These results are obtained from HAC1 variants that translated an active Hac1 protein from the un-spliced mRNA. Additionally, we show that the phosphatidylinositol-3-kinase Vps34 and the nutrient-sensing kinases TOR and GCN2 are key regulators of HAC1 mRNA translation and consequently the ER stress responses. Collectively, our data suggest that the cytosolic splicing and the translational de-repression of HAC1 mRNA are coordinated by unique and parallel networks of signaling pathways.

scholarly journals DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 90-96 ◽  
Author(s):  
T McClanahan ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Heat Shock ◽  
Shock Treatment ◽  
Northern Hybridization ◽  
Heat Shock Treatment ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Hsp70 Family ◽  
Transcriptional Start Sites

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation

1985 ◽  
Vol 5 (1) ◽  
pp. 17-26
Author(s):  
L Naumovski ◽  
G Chu ◽  
P Berg ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Calculated Molecular Weight ◽  
Base Pair Deletion ◽  
S1 Nuclease Mapping ◽  
Essential Function ◽  
Nuclease Mapping

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae

1986 ◽  
Vol 6 (10) ◽  
pp. 3401-3409
Author(s):  
D K Bishop ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmid Dna ◽  
Base Pair ◽  
Base Pairs ◽  
Plasmid Dnas ◽  
Repair Efficiency ◽  
Single Insertion ◽  
Base Pair Insertion

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

scholarly journals Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

2008 ◽  
Vol 190 (12) ◽  
pp. 4263-4271 ◽  
Author(s):  
Alexis I. Cocozaki ◽  
Ingrid R. Ghattas ◽  
Colin A. Smith
Keyword(s):  
Consensus Sequence ◽  
Point Mutations ◽  
Resonance Structure ◽  
Moderate Activity ◽  
Nuclear Magnetic Resonance Structure ◽  
Bacteriophage P22 ◽  
Base Pairs ◽  
Rna Hairpins ◽  
Transcription Antitermination ◽  
Theories Of Evolution

ABSTRACT Transcription antitermination in phages λ and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the λ N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxBleft complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and λ N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by λ N. Point mutations can dramatically alter boxB specificity between P22 and λ N. A boxB specific for P22 N can be mutated to λ N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

scholarly journals DNA SEQUENCES OF FRAMESHIFT AND OTHER MUTATIONS INDUCED BY ICR-170 IN YEAST

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 233-241
Author(s):  
Joachim F Ernst ◽  
D Michael Hampsey ◽  
Fred Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Genetic Analysis ◽  
Dna Sequences ◽  
Induced Mutations ◽  
Sequence Analyses ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Frameshift Mutations

ABSTRACT ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G·C additions at sites containing monotonous runs of three G·C base pairs. However, some (see PDF) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G·C base pairs.

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis

1988 ◽  
Vol 8 (9) ◽  
pp. 3703-3709
Author(s):  
T Tsukuda ◽  
S Carleton ◽  
S Fotheringham ◽  
W K Holloman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Consensus Sequence ◽  
Ustilago Maydis ◽  
Small Fragment ◽  
Direct Repeats ◽  
Base Pairs ◽  
Autonomously Replicating Sequence ◽  
Isolation And Characterization ◽  
Extrachromosomal Elements

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms

1986 ◽  
Vol 6 (11) ◽  
pp. 3847-3853
Author(s):  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Physical Structure ◽  
Base Pairs ◽  
Inducible Promoters ◽  
Upstream Promoter ◽  
Selection Of

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system

1988 ◽  
Vol 8 (6) ◽  
pp. 2442-2448 ◽  
Author(s):  
B Y Ahn ◽  
K J Dornfeld ◽  
T J Fagrelius ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Dna Sequences ◽  
Homologous Sequence ◽  
Insertion Mutation ◽  
Base Pairs ◽  
Recombination System ◽  
Plasmid Recombination ◽  
His3 Gene ◽  
Conversion Tract

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

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