Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Thursday Speaker Abstracts

CO-TEMPORAL FORCE AND FLUORESCENCE MEASUREMENTS REVEAL A RIBOSOMAL GEAR-SHIFT MECHANISM OF TRANSLATION REGULATION BY MRNA SECONDARY STRUCTURES Carlos Bustamante 1 ; Varsha Desai 1 ; Harry Noller 2 ; Laura Lancaster 2 ;

1 University of California, Berkeley, Berkeley, CA, USA 2 University of California, Santa Cruz, Santa Cruz, CA, USA

Ribosome translocation on mRNAs is often interrupted by secondary structures that represent mechanical barriers and that play a central role in translation regulation. Here, we investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G catalyzed translocation and is driven by the forward rotation of the small subunit head. Moreover, we modulate the magnitude of the hairpin barrier by force and surprisingly find that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold slower kinetic pathway prior to translocation. This shift into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub- codon steps, revealing a previously unobserved translocation intermediate. FUNCTIONAL PLASTICITY IN RING-SHAPED MOLECULAR MOTORS Shixin Liu 1 ; 1 Rockefeller University, Laboratory of Nanoscale Biophysics and Biochemistry, New York, NY, USA Ring ATPases represent a large and diverse group of molecular machines that couple their nucleotide hydrolysis activity to a mechanical task. I will discuss our single-molecule work on the eukaryotic replicative helicase CMG. Using correlative single-molecule fluorescence and force microscopy, we found that when uncoupled from a DNA polymerase, CMG opens a single- stranded (ss) DNA gate to traverse a forked junction and reside on double-stranded (ds) DNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA for continued fork progression. These results reveal unexpected plasticity in the CMG operation, enabling the ring motor to adapt to changing conditions and flexibly transition between distinct functional modes.

8