High-resolution landscape of an antibiotic binding site.
High-resolution landscape of an antibiotic binding site.
复制标题
DOI：
10.1038/s41586-023-06495-6
复制
发表时间：
2023-10
期刊：
影响因子：
64.8
通讯作者：
Nudler, Evgeny
中科院分区：
文献类型：
--
作者：
Yang, Kevin B.;Cameranesi, Maria;Gowder, Manjunath;Martinez, Criseyda;Shamovsky, Yosef;Epshtein, Vitaliy;Hao, Zhitai;Nguyen, Thao;Nirenstein, Eric;Shamovsky, Ilya;Rasouly, Aviram;Nudler, Evgeny
抗生素结合位点位于关键酶的重要区域，在耐药突变的背景下已经得到了广泛的研究；然而，它们的研究受到正选择的限制。利用多重基因组工程来克服这一限制，我们产生并鉴定了760个单残基突变体，涵盖了大肠杆菌RNA聚合酶(RNAP)的整个利福平结合位点。通过基因图谱药物-酶的相互作用，我们确定了一个阿尔法螺旋，其中突变显著增强或破坏利福平结合。我们在这个区域发现了延长抗生素结合时间的突变，通过诱导致命的DNA断裂将利福平从抑菌药物转化为杀菌药物。后者是复制依赖的，表明利福平通过在启动子上引起有害的转录-复制冲突而杀死。我们还发现了额外的结合位点突变，这些突变大大提高了RNAP的速度。快速RNAP耗尽了细胞中的核苷酸，改变了细胞对不同抗生素的敏感性，并提供了冷生长优势。最后，通过对天然rpoB序列多样性的作图，我们发现在自然界中经常发生改变RNAP特性或导致耐药性的功能性利福平结合位点突变。产生并鉴定了一系列跨越利福平结合位点所有可能的替代的RNA-聚合酶突变体，从而增加了我们对抗生素作用机制和细菌生理学的理解。
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug–enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription–replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature. A collection of RNA polymerase mutants spanning all possible substitutions of the rifampicin binding site is generated and characterized, increasing our understanding of antibiotic mechanisms and bacterial physiology.
登录
查看更多内容
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
DOI：
10.1126/science.aad3292
发表时间：
2016-01-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Baym M;Stone LK;Kishony R
通讯作者：
Kishony R
影响因子：
64.5
作者：
Kohanski, Michael A.;Dwyer, Daniel J.;Collins, James J.
通讯作者：
Collins, James J.
DOI：
10.1083/jcb.119.3.493
发表时间：
1992-11
期刊：
The Journal of cell biology
影响因子：
--
作者：
Gavrieli Y;Sherman Y;Ben-Sasson SA
通讯作者：
Ben-Sasson SA