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  Molecular motions at the 5’ stem loop of U4 snRNA: Implications for U4/U6 snRNP assembly

Cojocaru, V. (2005). Molecular motions at the 5’ stem loop of U4 snRNA: Implications for U4/U6 snRNP assembly. PhD Thesis, Georg-August-Universität, Göttingen, Germany.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-E8D1-B Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-D893-C
Genre: Thesis

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 Creators:
Cojocaru, V.1, Author              
Affiliations:
1Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              

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Free keywords: snRNA; RNA; Computer Simulation
 Abstract: The human 15.5K protein binds to the 5' stem-loop of U4 snRNA (KtU4), promotes the assembly of the spliceosomal U4/U6 snRNP, and is required for the recruitment of the 61K protein and the 20/60/90K protein complex to the U4 snRNA. In the crystallographic structure of the 15.5K-U4 snRNA complex, the RNA fold belongs to the family of kink turn (K-turn) motifs. This motif has a kink in the phosphodiester backbone that causes a sharp turn in the RNA helix. Two stems are connected by a purine-rich internal asymmetric loop, containing a flipped out uridine and two tandem sheared G-A base pairs. The shorter stem is attached to an external pentaloop. Using molecular dynamics simulations, I showed that the folding of KtU4 is assisted by protein binding. Conformational transitions such as the inter-conversion between alternative purine stacking schemes, the loss of G-A base pairs, and the opening of the K-turn (k-e motion) occurred only in the free RNA. The simulations provided the first atomic details of K-turn dynamics and were in excellent agreement with experimental data obtained by chemically probing the RNA structure and from single molecule FRET studies. In the free RNA, the k-e motion was triggered both by loss of G-A base pairs in the internal loop and backbone flexibility in the stems. However, the loss of G-A base pairs alone was insufficient for achieving a large opening of the free RNA. Essential dynamics showed that the loss of G-A base pairs is correlated along the first mode but anticorrelated along the third mode with the k-e motion. Based on these findings, I conclude that G-A base pair formation occurs upon binding to the 15.5K protein, stabilizing a selective orientation of the stems. The external loop was not revealed in the crystallographic structure of the 15.5KKtU4 complex. In the simulations, it adopted a specific orientation which did not persist in the unbound RNA and did not form when the natively occurring external loop was replaced by different loops or by an extended helix. I propose that the lack of stacking interactions between the last base pair of the stem and the adjacent nucleotide in the external loop are important for the correct folding of the RNA and might play a role in the subsequent binding of the 61K protein to the U4 snRNA.

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Language(s): eng - English
 Dates: 2005-06-282005-06-28
 Publication Status: Accepted / In Press
 Pages: -
 Publishing info: Göttingen, Germany : Georg-August-Universität
 Table of Contents: -
 Rev. Method: -
 Degree: PhD

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