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

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

3-POS Board 3 Observing Translation Initiation of the Ribosome on rpsO Gene Transcript by Using smFRET Shu-Ya Chang , Jin-Der Wen. Institute of Molecular and Cellular Biology, National Taiwan Univercity, Taipei, Taiwan. Many mRNAs fold into secondary structures; however, their codons must be in single-stranded form to be translated. Previous research has revealed that the ribosome itself has helicase activity during the translation process. The rpsO gene transcript of Escherichia coli regulates its own translation through the 5’ untranslated region (5’ UTR), which can fold into a pseudoknot or a double-hairpin conformation. The two structures can be interchanged spontaneously, but the ribosome can only bind the pseudoknot to initiate translation. On the pseudoknot, only the Shine- Dalgarno (SD) sequence but not the AUG start codon is exposed, so the ribosome has to unwind part of the secondary structure to complete the initiation. However, it remains unclear in which stage the conformation is opened by the ribosome. In this study, we characterize the conformational change of the rpsO 5’ UTR in the presence of the 30S ribosomal subunit and initiator tRNA (charged formyl-methionine tRNA) by using single-molecule fluorescence resonance energy transfer (smFRET). Our results show that the population of the pseudoknot form is increased when 30S binds to the RNA. 30S would begin to search for the AUG start codon after binding to the SD sequence of the pseudoknot by partially unwinding the local structures. In the present of the initiator tRNA, 30S could completely unwind a stem of the pseudoknot and form the pre-initiation complex. These results demonstrate that the 30S ribosomal subunit alone can perform its helicase activity during the initiation stage. Board 4 Real-Time Observation of Single Macromolecular Rotation Using Gold Nanorods Wen-Hsuan Chang 1 , Edward Yeung 2 , Hung-Wen Li 1 . 1 National Taiwan University, Taipei, Taiwan, 2 Iowa State University, Ames, IA, USA. Gold nanoparticles have high absorption and scattering cross section. The typical issue of single molecule fluorescence experiments is that fluorescence dyes photobleach and limit the observation time. Scattering of gold nanoparticles do not photobleach, making them suitable for long-time observation and fast optical imaging due to large scattering cross section. In particular, scattering light of gold nanorods is highly polarized along the long axis, allowing the anisotropic detection. Using laser dark-field microscopy, we image individual gold nanorods labeled on DNA molecules to observe the rotation of single macromolecules. Sensitive measurement can be made by out-of-focus images of gold nanorods, and analyzing the defocused image using geometric analysis, with the angle precision determined to be about one-degree. We will present the application to study protein-DNA interactions at the single-molecule level. 4-POS

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