# Kinetic characterization of RNA synthesis catalyzed by the model hyperthermophilic archaeon Thermococcus kodakarensis RNA polymerase

## TL;DR

This paper studies RNA synthesis in a heat-loving archaeon and finds unique properties in its RNA polymerase compared to bacteria and eukaryotes.

## Contribution

The study provides the first detailed kinetic comparison of archaeal RNA polymerase with bacterial and eukaryotic polymerases.

## Key findings

- T. kodakarensis RNA polymerase forms highly stable transcription complexes at both ambient and high temperatures.
- NTP concentration does not significantly affect the archaeal RNA polymerase's nucleotide addition rate.
- The archaeal RNA polymerase employs distinct regulatory strategies despite structural conservation with other polymerases.

## Abstract

At the broadest level of taxonomy, living organisms are divided into the domains Archaea, Bacteria, and Eukarya. Despite the significant differences in cellular organization, metabolic processes, and native environments between the prokaryotic Bacteria and Archaea compared to eukaryotes, the essential biological process of RNA synthesis is generally conserved across all domains. Archaea are the progenitors of Eukarya, and the homology of the singular archaeal RNA polymerase (RNAP) and eukaryotic RNA polymerases—particularly eukaryotic RNAP II (Pol II)—highlights the common evolutionary ancestry that resulted in the modern division of transcription activities between at least three distinct eukaryotic RNAPs. While detailed kinetic evaluations of the activities of bacteria and eukaryotic RNAPs have revealed both universal and significant differences in kinetic elongation schemes, identical comparisons to archaeal-derived RNAPs have largely been absent. Here, we characterize the elongation properties of Thermococcus kodakarensis (T. k.) RNAP, a model hyperthermophilic archaeon, and compare these properties to previously characterized bacterial and eukaryotic RNAPs. We demonstrate that T. k. RNAP forms transcription elongation complexes even more stable than Pol II at ambient temperatures, and remarkably stable complexes at high temperatures, wherein this archaeon thrives. We surprisingly observed no significant impact of NTP concentration on the rate of nucleotide addition by the archaeal RNAP at multiple temperatures that uniquely distinguishes the archaeal RNAP from bacterial and eukaryotic RNAPs. Our results reveal how distinct regulatory strategies can be employed for the archaeal RNAP despite the overall highly conserved structure and cellular functions of multi-subunit RNAPs.

Accurate and timely regulation of gene expression is critical for survival under dynamic conditions in all living organisms. Control of transcription initiation and elongation rates is a key parameter for cellular fitness, and determination of the conserved and unique regulatory strategies that control RNA polymerase activities is of paramount importance. How RNA synthesis is catalyzed by archaeal RNA polymerases provides insight into unique and conserved regulatory strategies for survival at the limits of life.