Details

Autor(en) / Beteiligte

• Hopkins, Julia Frances

Titel

Secondary structure rearrangements in RNA and RNA-protein complexes

Erscheinungsjahr

2008

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• The formation of the correct structure is critical for the function of many RNAs. The formation of RNA secondary structure is also required for the regulation of many RNAs. Molecular beacons, which are fluorescent oligonucleotide probes that form a hairpin structure, were used to study the formation of RNA secondary structure. These probes are conjugated to both a fluorophore and a quencher and fluoresce only when bound to their complementary RNA or DNA target. Molecular beacons were demonstrated to be useful for examining the folding and unfolding of the Tetrahymena ribozyme. A beacon complementary to a sequence in the core of the ribozyme specifically detected a misfolded intermediate. Hybridization of the beacon under native conditions revealed a linear relationship between the free energy of unfolding and the magnesium concentration. It was found that the core of the ribozyme was structured in the absence of Mg < super > 2+ < /er > . The splicing of the Tetrahymena pre-rRNA was studied using beacons targeted to different regions in the exons and intron/ribozyme of the RNA. Dissociation of the spliced RNAs was monitored in real-time after splicing was initiated. The mechanism of RNA annealing by the Hfq protein, which facilitates base pairing between small regulatory RNAs and their target mRNAs, was studied using a beacon and target oligonucleotide. It was shown that Hfq increased the rate of hybridization of these two oligomers up to 100-fold. Hfq was also able to promote the hybridization of the beacon to the full-length sRNA, DsrA. Simulations of kinetic parameters suggested that Hfq is likely required to associate with both the beacon and target strand during the annealing process. The molecular beacons were useful probes of RNA secondary structure. The large increase in signal upon hybridization, as well as their specificity, allow them to detect structural changes in RNA populations in real-time. The beacons are also a simple system with which to test the function of an RNA-binding protein and they demonstrated that Hfq accelerates the annealing of RNA strands.