L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations

1987 ◽  
Vol 7 (1) ◽  
pp. 420-426
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Gradient Centrifugation ◽  
Minus Strand ◽  
Double Stranded Rna ◽  
Major Class ◽  
Maturation In Vitro ◽  
Cscl Density Gradient ◽  
Viruslike Particles

Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus- strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism. In this paper, we show that mutations in mak10 or the pet18 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.

scholarly journals L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

1987 ◽  
Vol 7 (1) ◽  
pp. 420-426 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Gradient Centrifugation ◽  
Minus Strand ◽  
Double Stranded Rna ◽  
Major Class ◽  
Maturation In Vitro ◽  
Cscl Density Gradient ◽  
Viruslike Particles

Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus- strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism. In this paper, we show that mutations in mak10 or the pet18 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

scholarly journals K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (9) ◽  
pp. 4807-4815 ◽  
Author(s):  
M J Schmitt ◽  
D J Tipper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytoplasmic Membrane ◽  
Yeast Cells ◽  
Cross Hybridization ◽  
Double Stranded Rna ◽  
Killer System ◽  
Dsrna Viruses ◽  
Viruslike Particles ◽  
Killer Virus

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

scholarly journals Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561 ◽  
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

scholarly journals K28, a unique double-stranded RNA killer virus of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (9) ◽  
pp. 4807-4815
Author(s):  
M J Schmitt ◽  
D J Tipper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytoplasmic Membrane ◽  
Yeast Cells ◽  
Cross Hybridization ◽  
Double Stranded Rna ◽  
Killer System ◽  
Dsrna Viruses ◽  
Viruslike Particles ◽  
Killer Virus

The double-stranded RNA (dsRNA) viruses of Saccharomyces cerevisiae consist of 4.5-kilobase-pair (kb) L species and 1.7- to 2.1-kb M species, both found in cytoplasmic viruslike particles (VLPs). The L species encode their own capsid protein, and one (LA) has been shown to encode a putative capsid-polymerase fusion protein (cap-pol) that presumably provides VLPs with their transcriptase and replicase functions. The M1 and M2 dsRNAs encode the K1 and K2 toxins and specific immunity mechanisms. Maintenance of M1 and M2 is dependent on the presence of LA, which provides capsid and cap-pol for M dsRNA maintenance. Although a number of different S. cerevisiae killers have been described, only K1 and K2 have been studied in any detail. Their secreted polypeptide toxins disrupt cytoplasmic membrane functions in sensitive yeast cells. K28, named for the wine S. cerevisiae strain 28, appears to be unique; its toxin is unusually stable and disrupts DNA synthesis in sensitive cells. We have now demonstrated that 4.5-kb L28 and 2.1-kb M28 dsRNAs can be isolated from strain 28 in typical VLPs, that these VLPs are sufficient to confer K28 toxin and immunity phenotypes on transfected spheroplasts, and that the immunity of the transfectants is distinct from that of either M1 or M2. In vitro transcripts from the M28 VLPs show no cross-hybridization to denatured M1 or M2 dsRNAs, while L28 is an LA species competent for maintenance of M1. K28, encoded by M28, is thus the third unique killer system in S. cerevisiae to be clearly defined. It is now amenable to genetic analysis in standard laboratory strains.

scholarly journals Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

2000 ◽  
Vol 74 (24) ◽  
pp. 11671-11680 ◽  
Author(s):  
T. A. M. Osman ◽  
C. L. Hemenway ◽  
K. W. Buck
Keyword(s):  
Rna Polymerase ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Central Core ◽  
Minus Strand ◽  
Stem Loop ◽  
Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

scholarly journals The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1059-1070
Author(s):  
Susie C Howard ◽  
Arelis Hester ◽  
Paul K Herman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Signaling Pathway ◽  
Elongation Factor ◽  
Ras Signaling ◽  
Elongation Step ◽  
Rna Polymerase Ii Transcription

Abstract The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

scholarly journals RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (10) ◽  
pp. 4433-4440 ◽  
Author(s):  
N Chiannilkulchai ◽  
R Stalder ◽  
M Riva ◽  
C Carles ◽  
M Werner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Stop Codon ◽  
Sequence Similarity ◽  
Start Codon ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Multicopy Plasmid

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

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