Folding mechanism of Azoarcus Intron Ribozyme
Gokhan Caliskan 1,2, Robert Briber 3, Ursula Perez-Salas 2, Deverajan Thirumalai 4, Sarah Woodson 1
1 T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD
2 NCNR, NIST, Gaithersburg, MD
3 Dept. of Materials Science and Engineering University of Maryland, College Park, MD
4 Institute For Physical Science and Technology, University of Maryland, College Park, MD
RNA is the only biomolecule with the capability of self replication like DNA and enzymatic activity like proteins at the same time. RNA’s molecular structure is very similar to DNA, which is a polynucleotide, while its globular conformation and relative flexibility is more similar to protein formed of amino acids. RNA requires the negative charges of the phosphate groups on the backbone to be neutralized, a phenomenon called “counterion condensation”, in order to collapse before adopting the native conformation in presence of cations like Mg2+. Although some analogies from protein science are valid, RNA folding should still be studied in its own terms. A fundamental understanding of the physics behind the RNA collapse will improve the knowledge on polyelectrolytes, and is a requisite for the design and manipulation of RNA, which is a strong candidate to be used in pharmaceutical industry and in biotechnology.
We used Small Angle X-Ray Scattering (SAXS) technique to observe the size of the Azoarcus Intron Ribozyme as a function of Mg2+ and temperature. Our analysis pointed to a two-state transition. This transition was strongly temperature dependent and became less cooperative with decreasing temperature. We studied the effect of tertiary interactions on the collapse transition by disrupting a tertiary interaction in our sample by a mutation. Our measurements pointed that the tertiary interactions play an important role in the stabilization of the RNA. In the mutated sample, the midpoint of the collapse transition considerably increased and the transition became less cooperative. The collapsed state was not as compact as that of the wild type ribozyme. The results show that tertiary interactions do form in the collapsed state, and they play an active role in stabilizing the collapsed state.
We used a polyelectrolyte model to describe the distance distribution in Azoarcus Intron. The persistence length (lp) for the folded state that is calculated to be 10.5 Å increases to 27 Å for the unfolded state. Surprisingly, with two simple parameters, namely Rg and lp, the model nicely fits our data. This shows that, although very complex in function, biomolecules might still obey very simple rules that we are already familiar with from other synthetic systems like polyelectrolytes.
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Category: Biology and Biotechnology