Optical Tweezers Analysis on Structural Dynamics of the Truncated Human Telomerase RNA Structure
Date Issued
2012
Date
2012
Author(s)
Chang, Cheng-Fu
Abstract
Virus-encoded proteins, including structural proteins and enzymes, are usually produced by -1 programmed ribosomal frameshifting. In this mechanism, the structures of the messenger RNA and the slippery sequences are required. Moreover, secondary structures of the viral mRNA usually exist in pseudoknots. Previous experiments have shown that -1 frameshifting efficiency is related to the stability of the pseudoknots.
In this project, I used hTR ΔU177, a modified pseudoknot structure derived from the human telomerase RNA. The pseudoknot can be mimicked by annealing a hairpin and an ssRNA oligo derived form hTR ΔU177 . The bimolecular pseudoknot has a reduced ability to induce -1 frameshifting as the original hTR ΔU177.
By using optical tweezers, I measured the unfolding force of a series of hTR ΔU177-related structures and analyzed the relationship between the structural stability and formation of the triplex. The unfolding force of the hairpin was increased from about 17pN to 21.5pN in the presence of the ssRNA oligo. Furthermore, I found that the unfolding force was affected by the 5’ unpaired sequence of the ssRNA oligo as well. No matter whether the 5’ unpaired sequence were truncated or mutated, the unfolding force was decreased from 21.5pN to 19pN. I assume that there are extra hydrogen bonds formed between the unpaired nucleotides of the ssRNA and the hairpin G-C stem on the hairpin.
Moreover, when the loop 2 sequence in the pseudoknot structure was mutated, the unfolding force dropped from 47pN to 41pN. All the results suggest that both the triplex and the loop 2 sequence (5’ unpaired nucleotides of the ssRNA oligo in the bimolecular pseudoknots) contribute significantly to the stabilities of the hTR ΔU177-related structures.
Subjects
RNA
hairpin
pseudoknot
base-triple
single-molecule
optical tweezers
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
index.html
Size
23.27 KB
Format
HTML
Checksum
(MD5):8f5e01cf017f40f49e35cdc09af1611b