Biophysical Society Thematic Meeting

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.

4-POS

Board 4

Real-Time Observation of Single Macromolecular Rotation Using Gold Nanorods

Wen-Hsuan Chang

, Edward Yeung

, Hung-Wen Li

National Taiwan University, Taipei, Taiwan,

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.