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.
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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
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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.
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This study was supported by a Grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (18K19191).
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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.
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Yamori, Y., Hamakawa, M., Hidese, R. et al. Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles. Amino Acids 52, 275–285 (2020). https://doi.org/10.1007/s00726-019-02745-y
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DOI: https://doi.org/10.1007/s00726-019-02745-y