Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

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Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2382-2391.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2382-2391

Author(s):

C A Kaiser ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Signal Sequence ◽

Mutant Gene ◽

Yeast Cells ◽

Molecular Weight Characteristic ◽

Cell Extracts ◽

Substitution Mutations ◽

Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

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Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.816-822.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 816-822

Author(s):

H J Himmelfarb ◽

E Maicas ◽

J D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Blot Hybridization ◽

Single Copy ◽

Suppressor Gene ◽

Yeast Cells ◽

In Vitro Translation ◽

Nonsense Suppression ◽

Synthesis Rate

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

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Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2382 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2382-2391 ◽

Cited By ~ 47

Author(s):

C A Kaiser ◽

D Botstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Signal Sequence ◽

Mutant Gene ◽

Yeast Cells ◽

Molecular Weight Characteristic ◽

Cell Extracts ◽

Substitution Mutations ◽

Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

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The Herpes Simplex Virus vhs Protein Induces Endoribonucleolytic Cleavage of Target RNAs in Cell Extracts

Journal of Virology ◽

10.1128/jvi.73.9.7153-7164.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7153-7164 ◽

Cited By ~ 100

Author(s):

Mabrouk M. Elgadi ◽

Christopher E. Hayes ◽

James R. Smiley

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Pseudorabies Virus ◽

Host Protein ◽

In Vitro Translation ◽

Rna Decay ◽

Translation System ◽

Cell Extracts ◽

Simplex Virus

ABSTRACT The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5′ cap or a 3′ poly(A) tail in the RNA substrate, requires Mg2+, and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5′ quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

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Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3717-3725.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3717-3725

Author(s):

M Kornuc ◽

R Altman ◽

D Harrich ◽

J Garcia ◽

J Chao ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Transcriptional Regulation ◽

Binding Sites ◽

Mammalian Cells ◽

Yeast Cells ◽

Binding Domains ◽

Cell Extracts ◽

Dnase I Footprinting ◽

Transactivator Protein

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

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Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5555-5560.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5555-5560

Author(s):

H Iida

Keyword(s):

Heat Treatment ◽

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Adenylate Cyclase ◽

Uv Light ◽

Yeast Cells ◽

Wild Type ◽

Coding Region ◽

Resistant Mutants ◽

Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

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Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5555 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5555-5560 ◽

Cited By ~ 41

Author(s):

H Iida

Keyword(s):

Heat Treatment ◽

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Adenylate Cyclase ◽

Uv Light ◽

Yeast Cells ◽

Wild Type ◽

Coding Region ◽

Resistant Mutants ◽

Type Strains

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.

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Isolation of the SUP45 omnipotent suppressor gene of Saccharomyces cerevisiae and characterization of its gene product.

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.816 ◽

1985 ◽

Vol 5 (4) ◽

pp. 816-822 ◽

Cited By ~ 43

Author(s):

H J Himmelfarb ◽

E Maicas ◽

J D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Blot Hybridization ◽

Single Copy ◽

Suppressor Gene ◽

Yeast Cells ◽

In Vitro Translation ◽

Nonsense Suppression ◽

Synthesis Rate

The Saccharomyces cerevisiae SUP45+ gene has been isolated from a genomic clone library by genetic complementation of paromomycin sensitivity, which is a property of a mutant strain carrying the sup45-2 allele. This plasmid complements all phenotypes associated with the sup45-2 mutation, including nonsense suppression, temperature sensitivity, osmotic sensitivity, and paromomycin sensitivity. Genetic mapping with a URA3+-marked derivative of the complementing plasmid that was integrated into the chromosome by homologous recombination demonstrated that the complementing fragment contained the SUP45+ gene and not an unlinked suppressor. The SUP45+ gene is present as a single copy in the haploid genome and is essential for viability. In vitro translation of the hybrid-selected SUP45+ transcript yielded a protein of Mr = 54,000, which is larger than any known ribosomal protein. RNA blot hybridization analysis showed that the steady-state level of the SUP45+ transcript is less than 10% of that for ribosomal protein L3 or rp59 transcripts. When yeast cells are subjected to a mild heat shock, the synthesis rate of the SUP45+ transcript was transiently reduced, approximately in parallel with ribosomal protein transcripts. Our data suggest that the SUP45+ gene does not encode a ribosomal protein. We speculate that it codes for a translation-related function whose precise nature is not yet known.

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Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.620-631.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 620-631

Author(s):

Y H Tsay ◽

G W Robinson

Keyword(s):

Saccharomyces Cerevisiae ◽

Genomic Library ◽

Essential Gene ◽

Single Copy ◽

Yeast Cells ◽

Upstream Region ◽

Ergosterol Biosynthesis ◽

Temperature Sensitive ◽

Coding Region ◽

Phosphomevalonate Kinase

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.

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