Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles
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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.
KeywordsBranched-chain polyamine Polyamine RNA polymerase Transcription Archaea Hyperthermophile
Branched-chain polyamine synthase A
High-performance liquid chromatography
Liquid chromatography–mass spectrometry
Poly-acrylamide gel electrophoresis
Sodium dodecyl sulfate
TATA-box binding protein
Transcription factor B
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.
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.
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.
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