Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In Pseudomonas aeruginosa, HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria but contains conserved charged amino acids in its two alpha helices that interact with the rRNA. Here, we characterized the function of HPF in P. aeruginosa by performing mutagenesis of the conserved residues and then assaying mutant HPF alleles for their ability to protect ribosome integrity of starved P. aeruginosa cells. The results show that HPF functionally tolerates point mutations in charged residues and in the conserved Y71 residue as well as a C-terminal truncation. Double and triple mutations of charged residues in helix 1 in combination with a Y71F substitution reduce HPF activity. Screening for single point mutations that caused impaired HPF activity identified additional substitutions in the two HPF alpha helices. However, alanine substitutions in equivalent positions restored HPF activity, indicating that HPF is tolerant to mutations that do not disrupt the protein structure. Surprisingly, heterologous HPFs from Gram-positive bacteria that have long C-terminal domains functionally complement the P. aeruginosa Δhpf mutant, suggesting that HPF may play a similar role in ribosome protection in other bacterial species. Collectively, the results show that HPF has diverged among bacteria and is tolerant to most single amino acid substitutions. The Y71 residue in combination with helix 1 is important for the functional role of HPF in ribosome protection during bacterial starvation and resuscitation of the bacteria from dormancy.

中文翻译：

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铜绿假单胞菌核糖体冬眠促进因子的功能表征。

冬眠促进因子 (HPF) 是一种核糖体辅助蛋白，可在细菌饥饿期间使核糖体失活。在铜绿假单胞菌中，HPF 在细胞休眠时保护核糖体的完整性。HPF 的序列在细菌中有所不同，但在其与 rRNA 相互作用的两个 α 螺旋中包含保守的带电氨基酸。在这里，我们通过对保守残基进行诱变来表征 HPF 在铜绿假单胞菌中的功能，然后分析突变 HPF 等位基因保护饥饿铜绿假单胞菌核糖体完整性的能力细胞。结果表明，HPF 在功能上可以容忍带电残基和保守的 Y71 残基中的点突变以及 C 端截断。螺旋 1 中带电残基的双重和三重突变与 Y71F 取代相结合降低了 HPF 活性。对导致 HPF 活性受损的单点突变的筛选确定了两个 HPF α 螺旋中的额外替换。然而，等效位置的丙氨酸替换恢复了 HPF 活性，表明 HPF 可以耐受不破坏蛋白质结构的突变。令人惊讶的是，来自革兰氏阳性菌的异源 HPF 具有长 C 端结构域在功能上与铜绿假单胞菌Δ hpf互补突变体，表明 HPF 可能在其他细菌物种的核糖体保护中发挥类似作用。总的来说，结果表明 HPF 在细菌之间存在差异，并且对大多数单一氨基酸替换具有耐受性。Y71 残基与螺旋 1 结合对于 HPF 在细菌饥饿和从休眠中复苏细菌期间核糖体保护中的功能作用很重要。