scholarly journals Transcription apparatus of the yeast killer DNA plasmids: Architecture, function, and evolutionary origin

2018 ◽  
Author(s):  
Michal Sýkora ◽  
Martin Pospíšek ◽  
Josef Novák ◽  
Silvia Mrvová ◽  
Libor Krásný ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Kluyveromyces Lactis ◽  
Evolutionary Origin ◽  
Structural Elements ◽  
Extrachromosomal Elements ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Killer Plasmid ◽  
Yeast Killer

ABSTRACTTranscription of extrachromosomal elements such as organelles, viruses, and plasmids is dependent on cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeastKluyveromyces lactiscontains killer DNA plasmids that bear putative non-canonical RNAP genes. Here, we describe the architecture and evolutionary origin of this transcription machinery. We show that the two RNAP subunits interactin vivo, and this complex interacts with another two plasmid-encoded proteins - the mRNA capping enzyme, and a putative helicase which interacts with plasmid-specific DNA. Further, we identify a promoter element that causes 5’ polyadenylation of plasmid-specific transcriptsviaRNAP slippage during transcription initiation, and structural elements that precede the termination sites. As a result, we present a first model of the yeast killer plasmid transcription initiation and intrinsic termination. Finally, we demonstrate that plasmid RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively.

scholarly journals Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries

2002 ◽  
Vol 1 (3) ◽  
pp. 448-457 ◽  
Author(s):  
Toshimitsu Takagi ◽  
Eun-Jung Cho ◽  
Rozmin T. K. Janoo ◽  
Vladimir Polodny ◽  
Yasutaka Takase ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Rna Polymerase Ii ◽  
Subunit Interactions ◽  
Pol Ii ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Enzyme Subunits ◽  
Carboxyl Terminal

ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1 +) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.

Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function

1982 ◽  
Vol 2 (4) ◽  
pp. 346-354
Author(s):  
H Bussey ◽  
W Sacks ◽  
D Galley ◽  
D Saville
Keyword(s):  
Molecular Weight ◽  
In Vitro Translation ◽  
Heat Curing ◽  
In The Wild ◽  
Toxin Secretion ◽  
Killer Plasmid ◽  
Yeast Killer ◽  
Immunity Function

M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only on M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-] phenotypes were examined. All mutants became K- R- sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.

scholarly journals Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

2008 ◽  
Vol 28 (12) ◽  
pp. 3979-3994 ◽  
Author(s):  
Lu Gao ◽  
David S. Gross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Silencing ◽  
Pol Ii ◽  
Initiation Factors ◽  
Lysine Deacetylase ◽  
Evolutionarily Conserved ◽  
Mrna Capping ◽  
Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

scholarly journals The mouse Balbiani body maintains primordial follicle quiescence via RNA storage

2020 ◽  
Author(s):  
Lei Lei ◽  
Kanako Ikami ◽  
Haley Abbott ◽  
Shiying Jin
Keyword(s):  
Ovarian Function ◽  
Follicular Development ◽  
Primordial Follicle ◽  
Primordial Follicles ◽  
Mrna Decapping ◽  
Balbiani Body ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Decapping Enzyme

AbstractIn mammalian females, the transition between quiescent primordial follicles and follicular development is critical for maintaining ovarian function and reproductive longevity. In primary oocytes of mouse quiescent primordial follicles, Golgi complexes are organized into a spherical structure, the Balbiani body. Here, we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1A. Proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological disassembly of the B-body triggers translation of stored mRNAs and activates primordial follicles in culture and in vivo mouse model. Thus, primordial follicle quiescence is maintained by the B-body, and translationally inactive B-body-stored mRNAs may be regulated by 5’-capping.

scholarly journals Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase

2003 ◽  
Vol 77 (13) ◽  
pp. 7300-7307 ◽  
Author(s):  
Nayanendu Saha ◽  
Stewart Shuman ◽  
Beate Schwer
Keyword(s):  
Vaccinia Virus ◽  
Genetic Model ◽  
Genetic System ◽  
In Vivo Studies ◽  
Methyltransferase Activity ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Capping Enzymes

ABSTRACT Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Δ cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Δ complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.

scholarly journals Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 504 ◽  
Author(s):  
Tomoaki Ogino ◽  
Todd J. Green
Keyword(s):  
Rabies Virus ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Antiviral Agents ◽  
L Protein ◽  
Mrna Capping ◽  
Capping Enzyme

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5′-capping, cap methylation, and 3′-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.

scholarly journals A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes

2000 ◽  
Vol 74 (12) ◽  
pp. 5486-5494 ◽  
Author(s):  
C. Kiong Ho ◽  
Alexandra Martins ◽  
Stewart Shuman
Keyword(s):  
Fusion Protein ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Mrna Capping ◽  
Rna Triphosphatase ◽  
Capping Enzyme ◽  
Capping Enzymes

ABSTRACT Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme—vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase—to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Δceg1Δ cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme—Glu37, Glu39, Arg77, Glu192, and Glu194—that are essential for γ phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.

scholarly journals Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function.

1982 ◽  
Vol 2 (4) ◽  
pp. 346-354 ◽  
Author(s):  
H Bussey ◽  
W Sacks ◽  
D Galley ◽  
D Saville
Keyword(s):  
Molecular Weight ◽  
In Vitro Translation ◽  
Heat Curing ◽  
In The Wild ◽  
Toxin Secretion ◽  
Killer Plasmid ◽  
Yeast Killer ◽  
Immunity Function

M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only on M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-] phenotypes were examined. All mutants became K- R- sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.

scholarly journals The Essential Interaction between Yeast mRNA Capping Enzyme Subunits Is Not Required for Triphosphatase Function In Vivo

2000 ◽  
Vol 20 (24) ◽  
pp. 9307-9316 ◽  
Author(s):  
Yasutaka Takase ◽  
Toshimitsu Takagi ◽  
Philip B. Komarnitsky ◽  
Stephen Buratowski
Keyword(s):  
Amino Acids ◽  
Rna Polymerase Ii ◽  
Physiological Role ◽  
Pol Ii ◽  
Mrna Capping ◽  
Rna Triphosphatase ◽  
Capping Enzyme ◽  
Carboxy Terminal

ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.

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