Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles.
Branched-chain polyamine stabilizes RNA polymerase at elevated temperatures in hyperthermophiles.
复制标题
支链多胺可在高温下稳定嗜热菌中的 RNA 聚合酶。
DOI：
10.1007/s00726-019-02745-y
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发表时间：
2020
期刊：
影响因子：
3.5
通讯作者：
Fujiwara S.
中科院分区：
文献类型：
--
作者：
Yamori Y;Hamakawa M;Hidese R;Fukuda M;Atomi H;Fukuda W;Fujiwara S.
支链多胺（BCPAs）是在（超）嗜热菌中发现的独特的聚阳离子。柯达热球菌在85 °C下生长最佳，通过BCPA合成酶A（BpsA）将脱羧的S-腺苷甲硫氨酸（dcSAM）氨丙基基团依次添加到亚精胺（SPD）上，产生BCPAN 4-双（氨丙基）亚精胺。T。kodakarensisbpsA缺失突变体（DBP 1）在93 °C或高于93 °C的温度下不生长，并且仅在过量细胞质SPD积累后的长滞后期后在90 °C下生长。这表明BCPA在较高温度下的细胞生长中起着重要作用，并提高了BCPA参与控制基因表达的可能性。为了检测BCPA对转录的影响，从另一个携带His标记的RNA聚合酶的缺失突变体DBP 4（RNAPDBP 4）中提取RNA聚合酶（RNAP）核心部分，并将其酶性质与野生型（WT）细胞（RNAPWT）的RNAP进行比较。LC-MS分析表明，从RNAPWT中检测到9种核糖体蛋白，但只有一种RNAPDBP 4。这些结果表明，BCPA增加RNAP和核糖体之间的连接，以实现有效的转录和翻译偶联。两种RNAP在80 °C下的体外转录活性最高，但RNAPDBP 4的比活性低于RNAPWT。加入SPD和BCPA后，两者都增加了RNAPDBP 4的转录活性;然而，BCPA的升高是在低10倍的浓度下实现的。BCPA的加入也保护RNAPDBP 4在90 °C下不受热失活。这些结果表明，BCPA通过在高温下稳定RNAP复合物来增加柯达伤寒沙门氏菌的转录活性。
Branched-chain polyamines (BCPAs) are unique polycations found in (hyper)thermophiles.Thermococcus kodakarensisgrows optimally at 85 °C and produces the BCPAN4-bis(aminopropyl)spermidine by sequential addition of decarboxylatedS-adenosylmethionine (dcSAM) aminopropyl groups to spermidine (SPD) by BCPA synthase A (BpsA). TheT. kodakarensisbpsAdeletion 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 anotherbpsAdeletion mutant, DBP4 (RNAPDBP4), which carried a His-taggedrpoL, 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 RNAPWTbut 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 RNAPDBP4was 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 RNAPDBP4against thermal inactivation at 90 °C. These results suggest that BCPA increases transcriptional activity inT. kodakarensisby stabilizing the RNAP complex at high temperatures.