Biophysical Society Thematic Meeting | Hamburg 2022

Biophysics at the Dawn of Exascale Computers

Wednesday Speaker Abstracts

DISSECTING STRUCTURE, FUNCTION AND DYNAMICS OF THE DEAD-BOX HELICASE EIF4A BY SINGLE-MOLECULE FRET MICROSCOPY: REGULATION OF TRANSLATION INITIATION THROUGH MODULATION OF KINETIC COMPETITION BETWEEN ALTERNATIVE CONFORMATIONAL CYCLES Alexandra Z Andreou 1 ; Ulf Harms 1 ; Dagmar Klostermeier 1 ; 1 University of Muenster, Biophysical Chemistry, Muenster, Germany Translation initiation is a tightly regulated step in eukaryotic protein biosynthesis. Deregulation leads to altered gene expression, tumor formation, and cancer. Initiation requires unwinding of secondary structures in the 5’-untranslated region (5’-UTR) of mRNAs by the DEAD-box helicase eIF4A, an RNA-dependent ATPase and ATP-dependent helicase. The activities of eIF4A are stimulated by the translation factors eIF4B and eIF4G, and are linked to switching of eIF4A between open and closed conformations. Single-molecule FRET by confocal microscopy on eIF4A in solution shows that eIF4B, eIF4G, and the RNA itself modulate eIF4A activity by promoting formation of the closed state. Single-molecule experiments on surface-immobilized eIF4A by total internal reflection microscopy reveals that eIF4B and eIF4G jointly stimulate eIF4A activities by accelerating its conformational changes. The RNA substrate also influences eIF4A conformational dynamics: Short RNAs or RNA duplexes with short 5’-single-stranded regions only partially stimulate the ATPase activity, and closing is rate-limiting for the conformational cycle. Longer RNAs or RNA duplexes with a longer 5’-single-stranded region maximally stimulate ATP hydrolysis and promote closing of eIF4A. However, the rate constants of unwinding do not correlate with the length of a single-stranded region preceding a duplex. Instead, they reach a maximum for RNA with a single-stranded region of only six nucleotides. We propose a kinetic partitioning model where eIF4B, eIF4G, and the 5’-UTR of the respective mRNA affect eIF4A activity and translation initiation by modulating its partitioning between alternative conformational cycles: futile cycles (ATP hydrolysis without RNA unwinding), unproductive cycles (neither ATP hydrolysis nor duplex unwinding), and productive cycles (ATP hydrolysis coupled to duplex unwinding). Computational modeling of the flux of eIF4A through the different conformational cycles should enable predictions of translation efficiencies of mRNAs.

MOLECULAR SIMULATIONS OF GRAM-NEGATIVE BACTERIAL CELL ENVELOPES: A COMPLEX PICTURE IS EMERGING Syma Khalid University of Southampton, United Kingdom No Abstract

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