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High Resolution Cryo-EM Structure of 70S Ribosomal IC Bound to ATP-Trapped EttA

AJAS · 2020

Tejasri Vijayakumar · New York Academy of Sciences, The

Overview

Recently, a novel eubacterial ABC-F protein, energy-dependent translation throttle A (EttA) has been identified as a regulator of the elongation cycle of protein synthesis, depending on ATP and ADP levels. The 70S pretranslocation (PRE) ribosomal complex exists in a spontaneous equilibrium between two global conformational states, called macrostates I and II (MS-I and MS-II). The transition from MS-I to MS-II is facilitated by the movement of the L1 stalk on the large subunit toward the small subunit, which is critical for the entry of the 70S initiation complex (70S IC) into the elongation cycle. The goal of this study is to gain insight on the structural interactions that enable EttA to gate ribosome entry into the translation elongation cycle. Dual glutamate-to-glutamine substitutions in the catalytic bases in both ABC domains (EttA-EQ2 mutant) prevent ATP hydrolysis and trap EttA in its ATP-bound conformation. In our study, we used a non-enzymatically assembled 70S IC containing initiator tRNAfMet and ATP-trapped EttA-EQ2 to obtain cryo-EM micrographs. We performed initial model-building steps in Relion-3.0 and further processed the data in CryoSPARC. With these softwares, we constructed high-resolution 3D maps ranging from 3.28 Å to 4.28 Å. Although previous cryo-EM studies of the EttA-EQ2-bound PRE complex generated a major class at 7.5 Å, this is the first time a high-resolution map has been generated. Our results, apart from providing important insights into the molecular interactions that mediate the function of EttA as a translational regulator, also help us understand the drug resistant activity of bacterial ABC-F family members that confer resistance to widely used ribosome-inhibiting antibiotics.

Competition history

  • AJAS 2020 Category not listed

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Source: AAAS Annual Meeting (Confex) / American Junior Academy of Science

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