Mechanism of action of ABCF ATPases during translation

在细胞中，蛋白质的合成发生在核糖体上，并由大量专门的翻译因子促进。除了翻译GTP酶之外，还存在通过翻译ATP酶调节翻译的新兴作用，例如真核生物中的延伸因子EF 3和细菌中的抗生素抗性（ARE）ABCF ATP酶。在真核生物中，EF 3在翻译延伸期间与核糖体相互作用，在那里它促进E-tRNA的释放。也有报道称，在某些条件下，eEF 3也可以促进核糖体分裂。10多年前，仅报道了体外重组酵母EF 3 - 80 S复合物的单一低（9.9 μ m）分辨率结构。此外，酵母和真菌有许多eEF 3的同源物，如New 1 p，但目前还不清楚它们是否也参与翻译。在这里，我们建议使用体外和体内方法来确定EF 3和New 1 p与80 S核糖体复合的结构。这些研究对于理解为什么酵母和真菌需要这种因子才能生存，以及这种功能是否被高等真核生物中的另一种因子所取代是必要的。ARE-ABCF ATP酶存在于革兰氏阳性产芽孢杆菌（如枯草芽孢杆菌）以及致病菌（如葡萄球菌、链球菌和肠球菌）中。ARE-ABCF蛋白赋予对在核糖体的肽基转移酶中心（PTC）处或附近结合的抗生素的抗性，其中不同的ARE-ABCF蛋白具有不同的抗生素特异性。到目前为止，还没有与核糖体复合的蛋白质的ARE-ABCF亚家族的结构，因此，仍然不清楚这些蛋白质如何与核糖体结合以及它们如何机械地赋予对特定核糖体靶向抗生素类的抗性。在这里，我们建议确定与核糖体复合的ARE-ABCF蛋白质的结构，以了解这些蛋白质如何与核糖体结合，以及它们如何在机械上赋予对特定核糖体靶向抗生素类的抗性。鉴于多重耐药细菌的不断出现，了解这些新的细菌耐药机制对于开发新的改进的抗生素将是重要的。

In cells, the synthesis of proteins occurs on ribosomes and is facilitated by a plethora of specialized translation factors. In addition to translational GTPases, there is an emerging role for regulation of translation via translational ATPases, such as the elongation factor EF3 in eukaryotes and the antibiotic resistance (ARE) ABCF ATPases in bacteria. In eukaryotes, EF3 interacts with the ribosome during translation elongation, where it promotes release of the E-tRNA. There are also reports that under some conditions eEF3 can also promote ribosomal splitting. Only a single low (9.9 Å) resolution structure of an in vitro reconstituted yeast EF3-80S complex has been reported from over 10 years ago. Additionally, Yeast and fungi have many homologues of eEF3, such as New1p, however, it remains unclear whether they are also involved in translation. Here we propose to determine structures of EF3 and New1p in complex with the 80S ribosome using both in vitro and in vivo approaches. Such investigations will be necessary to understand why yeast and fungi require this factor for survival, and whether this function is taken over by another factor in higher eukaryotes. The ARE-ABCF ATPases are found in Gram-positive antibiotic-producing bacteria, such as Bacillus subtilis, as well as in pathogenic bacteria, such as Staphylococcus, Streptococcus, and Enterococcus. The ARE-ABCF proteins confer resistance to antibiotics that bind at or near the peptidyl-transferase center (PTC) of the ribosome, with different ARE-ABCF proteins having different antibiotic specificities. To date, there are no structures of the ARE-ABCF subfamily of proteins in complex with the ribosome, therefore, it remains unclear how these proteins bind to the ribosome and mechanistically how they confer resistance to the specific ribosome-targeting antibiotic classes. Here we propose to determine structures of ARE-ABCF proteins in complex with the ribosome to understand how these proteins bind to the ribosome and mechanistically how they confer resistance to the specific ribosome-targeting antibiotic classes. Given the ever-increasing emergence of multi drug resistant bacteria, understanding these novel bacterial resistance mechanisms will be important for development of new improved antibiotics.