Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

6-POS Board 6 Observing the Helicase Activity of Ribosome 30S during Translation Initiation by Using Optical Tweezers Yi-Lan Chen 1 , Jin-Der Wen 2 . 1 Genome and Systems Biology Program, National Taiwan University, Taipei, Taiwan, 2 Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan. In prokaryotes, recognition of ribosomal binding site (RBS) which includes the Shine-Dalgarno sequence (SD) and AUG start codon is important for translation initiation. Structured 5’-UTR of mRNA might influence the protein expression. On the other hand, it has been proposed that ribosomal proteins S3 and S4 in the 30S are important to the helicase activity of the ribosome. This implies that the 30S itself might open the structured 5’-UTR during translation initiation. We test this hypothesis by using optical tweezers. When we added the 30S to bind the SD sequence before a hairpin, the unfolding force of the hairpin became lower. This suggests that the 30S itself interacts with and destabilizes the hairpin. Furthermore, the 30S needs the initiator tRNA (fmet-tRNA) to dock properly on RBS. Therefore, we added both fmet-tRNA and the 30S to form pre-initiation complex. We found 30S was correctly initiated at the start codon. This result might explain how translation initiation happens and how 30S interacts with structured 5’- UTR. We propose that the 30S binds to SD first. Then, the 30S searches and partially opens surrounding structures. Finally, it will accommodate at the correct site after fmet-tRNA pairs to the AUG start codon. With more control experiments in the near future, we wish this study will help us to understand how the 30S and structured 5’-UTR cooperate to achieve the regulation of translation initiation.

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