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A comparison of <i>Dictyostelium discoideum</i> 3'-5' RNA polymerases reveals a conserved tRNA<sup>His</sup> guanylyltransferase residue that plays a dual role in catalysis.

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Metal Ion Requirement for Catalysis by 3'-5' RNA Polymerases.

Brandon W J Iwaniec1,2, Madison M Allegretti2, Jane E Jackman1,2

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This study reveals that Thg1-like proteins (TLPs) utilize diverse divalent metal ions for catalysis, impacting nucleotide addition. Different TLPs show unique metal ion dependencies, with some metals causing mutations, similar to polymerases.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • The two-metal ion mechanism is key for 5'-3' polymerase activity.
  • 3'-5' polymerases, like Thg1 and TLPs, add nucleotides to RNA 5'-ends but their metal ion requirements are less understood.
  • Previous studies lacked detailed analysis of divalent cations beyond magnesium for TLP catalysis.

Purpose of the Study:

  • To investigate the effects of five divalent cations (Mg2+, Mn2+, Co2+, Ni2+, Ca2+) on TLP-catalyzed nucleotide addition and 5'-activation.
  • To characterize the distinct metal ion dependencies of TLPs from diverse organisms.
  • To elucidate the role of the two-metal ion mechanism in TLP catalysis.

Main Methods:

  • Recombinant purification of metal-free TLPs.
  • Assays for templated nucleotide addition activity.
  • Kinetic analysis of 5'-activation by ATP.
  • Comparative analysis of divalent cation effects across different TLP species.

Main Results:

  • TLPs exhibit distinct divalent metal ion concentration and identity dependencies for catalysis.
  • Metal ion usage patterns in TLPs share features with both 5'-3' polymerases and DNA/RNA ligases.
  • Some metal ions were mutagenic in TLP-catalyzed reactions, mirroring 5'-3' polymerase behavior.
  • Direct evidence shows both ATP and the incoming NTP occupy the active site during 5'-adenylylation.

Conclusions:

  • Divalent metal ions play a critical and varied role in TLP catalysis.
  • TLPs employ a two-metal ion mechanism with unique characteristics.
  • This study provides the first in-depth mechanistic insights into TLP metal ion utilization.