Mechanism of action of ABCF ATPases during translation

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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.