scholarly journals Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant

Virology ◽  
2016 ◽  
Vol 487 ◽  
pp. 27-40
Author(s):  
Jessica Tate ◽  
Rachel L. Boldt ◽  
Baron D. McFadden ◽  
Susan M. D’Costa ◽  
Nicholas M. Lewandowski ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Temperature Sensitive ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Virus Mutant

scholarly journals mRNA Capping Enzyme Activity Is Coupled to an Early Transcription Elongation

2004 ◽  
Vol 24 (14) ◽  
pp. 6184-6193 ◽  
Author(s):  
Hye-Jin Kim ◽  
Seok-Ho Jeong ◽  
Jeong-Hwa Heo ◽  
Su-Jin Jeong ◽  
Seong-Tae Kim ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
State Level ◽  
Molecular Defect ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Nascent Transcript ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Early Transcription

ABSTRACT One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.

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.

scholarly journals Thermodynamics of ligand binding by the yeast mRNA-capping enzyme reveals different modes of binding

2004 ◽  
Vol 384 (2) ◽  
pp. 411-420 ◽  
Author(s):  
Isabelle BOUGIE ◽  
Amélie PARENT ◽  
Martin BISAILLON
Keyword(s):  
Ligand Binding ◽  
Entropy Change ◽  
Enthalpy Change ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Eukaryotic Mrnas ◽  
Entropic Effect ◽  
A Minor ◽  
Rna Capping ◽  
Capping Enzymes

RNA-capping enzymes are involved in the synthesis of the cap structure found at the 5′-end of eukaryotic mRNAs. The present study reports a detailed study on the thermodynamic parameters involved in the interaction of an RNA-capping enzyme with its ligands. Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates. Our thermodynamic analyses indicate that the initial association of GTP with the Ceg1 protein is driven by a favourable enthalpy change (ΔH=−80.9 kJ/mol), but is also clearly associated with an unfavourable entropy change (TΔS=−62.9 kJ/mol). However, the interaction between Ceg1 and RNA revealed a completely different mode of binding, where binding to RNA is clearly dominated by a favourable entropic effect (TΔS=20.5 kJ/mol), with a minor contribution from a favourable enthalpy change (ΔH=−5.3 kJ/mol). Fluorescence spectroscopy also allowed us to evaluate the initial binding of GTP to such an enzyme, thereby separating the GTP binding step from the concomitant metal-dependent hydrolysis of GTP that results in the formation of a covalent GMP–protein intermediate. In addition to the determination of the energetics of ligand binding, our study leads to a better understanding of the molecular basis of substrate recognition by RNA-capping enzymes.

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