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

Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles. Thermococcus kodakarensis grows optimally at 85 °C and produces the BCPA N 4 -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 (RNAP DBP4 ), which carried a His-tagged rpoL , and its enzymatic properties were compared with those of RNAP from wild-type (WT) cells (RNAP WT ). LC–MS analysis revealed that nine ribosomal proteins were detected from RNAP WT but only one form RNAP DBP4 . 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 RNAP DBP4 was lower than that of RNAP WT . Upon addition of SPD and BCPA, both increased the transcriptional activity of RNAP DBP4 ; however, elevation by BCPA was achieved at a tenfold lower concentration. Addition of BCPA also protected RNAP DBP4 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.