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Literature summary for 3.1.13.B1 extracted from

  • Park, J.; Kang, M.; Kim, M.
    Unraveling the mechanistic features of RNA polymerase II termination by the 5'-3' exoribonuclease Rat1 (2015), Nucleic Acids Res., 43, 2625-2637 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.13.B1

Activating Compound

Activating Compound Comment Organism Structure
ATP ATP significantly enhances Rat1/Rai1-mediated RNAPII termination. The ATP-dependent effect seems to be unique to Rat1/Rai1. ATP addition does not stimulate the processivity of Rat1. Rather, it slightly reduces the exonuclease activity because 1 mM of ATP is sufficient to chelate the Mg2+ ion necessary for the nuclease activity of Rat1, suggesting that ATP affects Rat1-mediated termination in a somewhat different manner Saccharomyces cerevisiae
additional information NTP misincorporation induces RNAPII pausing and enhances termination by Rat1/Rai1. When ATP misincorporates into RNA transcripts, the elongation of RNAPII is significantly blocked, presumably due to the disruption and/or rearrangement of the RNAPII active center, RNAPII with a disrupted active center might be more effectively terminated by Rat1/Rai1 Saccharomyces cerevisiae

Cloned(Commentary)

Cloned (Comment) Organism
recombinant overexpression of His6-tagged Rat1 in Escherichia coli strain BL21 Codon-Plus(DE3)RIL. The recombinant Rat1/Rai1 cannot terminate Escherichia coli RNAP in vitro and addition of each NTP has no effect either Saccharomyces cerevisiae

Protein Variants

Protein Variants Comment Organism
E203A/D233A/D235A site-directed mutagenesis, mutation of three active site residues, mutant rat1EDD does not show 5'-3' exoribonuclease activity, and rat1EDD/Rai1 complex does not degrade RNAs or decrease RNAPII elongation, demonstrating that RNA degradation by exonuclease activity is critical to RNAPII termination Saccharomyces cerevisiae

Inhibitors

Inhibitors Comment Organism Structure
EDTA
-
 Saccharomyces cerevisiae

Localization

Localization Comment Organism GeneOntology No. Textmining
nucleus
-
 Saccharomyces cerevisiae 5634
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ absolutely required for catalysis Saccharomyces cerevisiae

Organism

Organism UniProt Comment Textmining
Saccharomyces cerevisiae Q02792
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged Rat1 from Escherichia coli strain BL21 Codon-Plus (DE3) RIL by nickel affinity chromatography and anion exchange chromatography, followed by gel filtration Saccharomyces cerevisiae

Reaction

Reaction Comment Organism Reaction ID
exonucleolytic cleavage in the 5'- to 3'-direction to yield nucleoside 5'-phosphates cleavage scheme, Rail/Rat1 mechanism and mechanism of RNAPII termination, overview Saccharomyces cerevisiae
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information nuclear yeast Rat1 catalyzes exonucleolytic cleavage of RNA in the 5'- to 3'-direction to yield nucleoside 5'-phosphates, it forms a complex with Rai1 that stabilizes Rat1 and helps target 5x02 monophosphate RNA by its diphosphohydrolase activity. The efficiency of termination increases as the RNA transcript undergoing degradation by Rat1 gets longer, which suggests that Rat1 may generate a driving force for dissociating RNAPII from the template while degrading the nascent transcripts to catch up to the polymerase. Rat1/Rai1 itself is not sufficient to terminate RNAPII in vitro. Yeast Rat1/Rai1 does not terminate Escherichia coli RNAP, implying that a specific interaction between Rat1/Rai1 and RNAPII may be required for termination. Rat1/Rai1 do not show ATPase activity, ATP hydrolysis may be crucial to promote RNAPII termination but is not driven by Rat1/Rai1. The 5'-3' exoribonuclease activity of Rat1 is essential for RNAPII termination, quantification of the remaining RNAs after Rat1/Rai1 treatment without or with ATP Saccharomyces cerevisiae ?
-
 ?

Synonyms

Synonyms Comment Organism
5'-3' exoribonuclease 2 UniProt Saccharomyces cerevisiae
Rat1
-
 Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Saccharomyces cerevisiae

General Information

General Information Comment Organism
evolution yeast Rat1 is evolutionarily well conserved from yeast to human (Xrn2 in human) Saccharomyces cerevisiae
physiological function yeast Rat1 is an essential nuclear protein. Within a complex with Rai1, the 5'-3' exoribonuclease Rat1 promotes termination of RNA polymerase II (RNAPII) on protein-coding genes. The 5'-3' exoribonuclease activity of Rat1 is essential for RNAPII termination. Rat1 forms a complex with Rai1 that stabilizes Rat1 and helps target 5' monophosphate RNA by its diphosphohydrolase activity. Within a complex with Rai1, the 5'-3' exoribonuclease Rat1 promotes termination of RNAPII on protein-coding genes, overview. RNAPII is prone to more effective termination by Rat1/Rai1 when its catalytic site is disrupted due to NTP misincorporation, implying that paused RNAPII, which is often found in vivo near termination sites, can adopt a similar configuration to Rat1/Rai1 and trigger termination. Rat1/Rai1 itself is not sufficient to terminate RNAPII in vitro. Multiple-mechanistic features contribute to Rat1-mediated termination of RNAPII. NTP misincorporation induces RNAPII pausing and enhances termination by Rat1/Rai1, Rat1/Rai1 more effectively terminates RNAPII when a non-cognate NTP (ATP, CTP or UTP), rather than cognate GTP. Exoribonuclease activity is required for Rat1 not only to approach RNAPII but also to accumulate a sufficient driving force to dislodge the polymerase from the DNA template Saccharomyces cerevisiae