Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.
The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.
Woźniak
Anna K
AK
Heinrich-Heine-Universität Düsseldorf, Institut für molekulare Physikalische Chemie, 40225 Düsseldorf, Germany.
Nottrott
Stephanie
S
Kühn-Hölsken
Eva
E
Schröder
Gunnar F
GF
Grubmüller
Helmut
H
Lührmann
Reinhard
R
Seidel
Claus A M
CA
Oesterhelt
Filipp
F
D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006834:Hydrazines; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D015347:RNA Probes; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D017381:Spliceosomes / Q000378:metabolism
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D015347:RNA Probes; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C405482:U4 small nuclear RNA; D012313:RNA; D012964:Sodium; D008274:Magnesium