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Amino Acids

pp 1–11 | Cite as

Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles

  • Yuka Yamori
  • Masafumi Hamakawa
  • Ryota Hidese
  • Moeko Fukuda
  • Haruyuki Atomi
  • Wakao Fukuda
  • Shinsuke FujiwaraEmail author
Original Article
Part of the following topical collections:
  1. Polyamines: Biochemical and Pathophysiological Properties

Abstract

Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles. Thermococcus kodakarensis grows optimally at 85 °C and produces the BCPA N4-bis(aminopropyl)spermidine by sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine (SPD) by BCPA synthase A (BpsA). The T. kodakarensis bpsA deletion mutant (DBP1) did not grow at temperatures at or above 93 °C, and grew at 90 °C only after a long lag period following accumulation of excess cytoplasmic SPD. This suggests that BCPA plays an essential role in cell growth at higher temperatures and raises the possibility that BCPA is involved in controlling gene expression. To examine the effects of BCPA on transcription, the RNA polymerase (RNAP) core fraction was extracted from another bpsA deletion mutant, DBP4 (RNAPDBP4), which carried a His-tagged rpoL, and its enzymatic properties were compared with those of RNAP from wild-type (WT) cells (RNAPWT). LC–MS analysis revealed that nine ribosomal proteins were detected from RNAPWT but only one form RNAPDBP4. These results suggest that BCPA increases the linkage between RNAP and ribosomes to achieve efficient coupling of transcription and translation. Both RNAPs exhibited highest transcription activity in vitro at 80 °C, but the specific activity of RNAPDBP4 was lower than that of RNAPWT. Upon addition of SPD and BCPA, both increased the transcriptional activity of RNAPDBP4; however, elevation by BCPA was achieved at a tenfold lower concentration. Addition of BCPA also protected RNAPDBP4 against thermal inactivation at 90 °C. These results suggest that BCPA increases transcriptional activity in T. kodakarensis by stabilizing the RNAP complex at high temperatures.

Keywords

Branched-chain polyamine Polyamine RNA polymerase Transcription Archaea Hyperthermophile 

Abbreviations

AA

Amino acids

ASW

Artificial seawater

BCPA

Branched-chain polyamine

BpsA

Branched-chain polyamine synthase A

Ca(OAc)2

Calcium acetate

DTT

Dithiothreitol

EtBr

Ethidium bromide

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC

High-performance liquid chromatography

KOAc

Potassium acetate

LC–MS

Liquid chromatography–mass spectrometry

LCPA

Long-chain polyamine

Mg(OAc)2

Magnesium acetate

PA

Polyamine

PAGE

Poly-acrylamide gel electrophoresis

RNAP

RNA polymerase

SDS

Sodium dodecyl sulfate

SPD

Spermidine

TBP

TATA-box binding protein

TCA

Trichloroacetic acid

TFB

Transcription factor B

Tris

Tris(hydroxymethyl)aminomethane

Notes

Acknowledgements

This study was mainly supported by a grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (18K19191). Bioinformatic analysis was supported by a Grant for Individual Special Research, provided by Kwansei-Gakuin University.

Author contributions

All the authors contributed to study design. YY, MH, RH, MF, and SF performed the experiments. YY, FW, HA, and SF wrote the manuscript. All authors reviewed and approved the final draft.

Funding

This study was supported by a Grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (18K19191).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

726_2019_2745_MOESM1_ESM.docx (19 kb)
Supplementary file1 (DOCX 19 kb)
726_2019_2745_MOESM2_ESM.docx (21 kb)
Supplementary file2 (DOCX 21 kb)
726_2019_2745_MOESM3_ESM.docx (16 kb)
Supplementary file3 (DOCX 16 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Bioscience, Graduate School of Science and TechnologyKwansei-Gakuin UniversitySandaJapan
  2. 2.Department of Synthetic Chemistry and Biological Chemistry, Graduate School of EngineeringKyoto UniversityKyotoJapan

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