The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II

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.

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A55 Molecular systematics of sturgeon nucleocytoplasmic large DNA viruses

Virus Evolution ◽

10.1093/ve/vez002.054 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

S Clouthier ◽

E Anderson ◽

G Kurath ◽

R Breyta

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Small Subunit ◽

Sequence Length ◽

Dna Viruses ◽

Mrna Capping ◽

Capping Enzyme ◽

Nucleocytoplasmic Large Dna Virus ◽

Nucleocytoplasmic Large Dna Viruses

Abstract Namao virus (NV) is a sturgeon nucleocytoplasmic large DNA virus (sNCLDV) that can cause a lethal disease of the integumentary system in lake sturgeon Acipenser fulvescens. As a group, the sNCLDV have not been assigned to any currently recognized taxonomic family of viruses. In this study, a dataset of NV DNA sequences was generated and assembled as two non-overlapping contigs of 306 and 448 base pairs (bp) and then used to conduct a comprehensive systematics analysis using Bayesian phylogenetic inference for NV, other sNCLDV, and representative members of six families of the NCLDV superfamily. The phylogeny of NV was reconstructed using protein homologues encoded by nine nucleocytoplasmic virus orthologous genes (NCVOGs): NCVOG0022—mcp, NCVOG0038—DNA polymerase B elongation subunit, NCVOG0076—VV A18-type helicase, NCVOG0249—VV A32-type ATPase, NCVOG0262—AL2 VLTF3-like transcription factor, NCVOG0271—RNA polymerase II subunit II, NCVOG0274—RNA polymerase II subunit I, NCVOG0276—ribonucleotide reductase small subunit, and NCVOG1117—mRNA capping enzyme. The accuracy of our phylogenetic method was evaluated using a combination of Bayesian statistical analysis and congruence analysis. Stable tree topologies were obtained with datasets differing in target molecule(s), sequence length, and taxa. Congruent topologies were obtained in phylogenies constructed using individual protein datasets and when four proteins were used in a concatenated approach. The major capsid protein phylogeny indicated that ten representative sNCLDV form a monophyletic group comprised of four lineages within a polyphyletic Mimi-Phycodnaviridae group of taxa. Overall, the analyses revealed that Namao virus is a member of the Mimiviridae family with strong and consistent support for a clade containing NV and CroV as sister taxa.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00019-08 ◽

2008 ◽

Vol 28 (12) ◽

pp. 3979-3994 ◽

Cited By ~ 34

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.

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mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain

Genes & Development ◽

10.1101/gad.11.24.3319 ◽

1997 ◽

Vol 11 (24) ◽

pp. 3319-3326 ◽

Cited By ~ 305

Author(s):

E.-J. Cho ◽

T. Takagi ◽

C. R. Moore ◽

S. Buratowski

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Mrna Capping ◽

Capping Enzyme ◽

Carboxy Terminal

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Identification of novel RNA Polymerase II CTD interaction sites on the mRNA Capping Enzyme

10.1101/2020.02.25.964700 ◽

2020 ◽

Author(s):

Marcus G. Bage ◽

Rajaei Almohammed ◽

Victoria H. Cowling ◽

Andrei V. Pisliakov

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Export ◽

Enhanced Sampling ◽

Interaction Sites ◽

Mrna Capping ◽

Capping Enzyme ◽

Dynamics Simulations ◽

Computational Analyses ◽

Rna Polymerase Ii Ctd

1AbstractRecruitment of the mRNA Capping Enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5’ mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterisation of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE surface and quantify their relative contributions to CTD binding. We also identify differences in the CTD binding conformation when phosphorylated at either the Serine-2 or Serine-5 positions, thus providing insights into how the CE reads the CTD code. The computational findings are then validated by binding and activity assays. These novel CTD interaction sites are compared with cocrystal structures of the CE-CTD complex in different eukaryotic taxa, leading to the conclusion that this interface is considerably more conserved than previous structures have indicated.

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