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Bacterial RNA Polymerase00:43

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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
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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

  • 1Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, 43210.

Biorxiv : the Preprint Server for Biology
|April 1, 2025
PubMed
Summary

Thg1-like proteins (TLPs) use diverse metal ions for catalysis, unlike canonical polymerases. This study reveals distinct metal ion preferences and mutagenic effects in TLPs, offering new insights into their unique two-metal ion mechanism.

Keywords:
TLPThg1adenylylationdivalent cationstRNAtwo-metal ion mechanism

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • The two-metal ion mechanism is key for 5'-3' polymerase activity.
  • Thg1-like proteins (TLPs) are 3'-5' polymerases with a similar reaction but limited understanding of their metal ion requirements.

Purpose of the Study:

  • To investigate the effects of various divalent metal ions on TLP templated nucleotide addition and 5'-adenylylation.
  • To characterize the distinct metal ion dependencies and catalytic mechanisms of TLPs.

Main Methods:

  • Recombinant purification of metal-free TLPs from diverse organisms.
  • Assaying templated nucleotide addition and 5'-activation kinetics with five different divalent cations (Mg2+, Mn2+, Co2+, Ni2+, Ca2+).

Main Results:

  • TLPs exhibit unique and diverse dependencies on divalent metal ion identity and concentration for catalysis.
  • Metal ion usage patterns in TLPs share characteristics with both 5'-3' polymerases and DNA/RNA ligases.
  • Some metal ions were observed to be mutagenic in TLP catalysis, and direct evidence for simultaneous ATP and substrate NTP in the active site was found.

Conclusions:

  • TLPs utilize a flexible two-metal ion mechanism with distinct metal ion preferences compared to canonical polymerases.
  • This study provides the first in-depth analysis of the two-metal ion mechanism in TLP catalysis, revealing novel aspects of their enzymatic function.