We present a systematic study of the long-timescale dynamics of the Drew-Dickerson dodecamer (DDD: d(CGCGAATTGCGC)2) a prototypical B-DNA duplex. Using our newly parameterized PARMBSC1 force field, we describe the conformational landscape of DDD in a variety of ionic environments from minimal salt to 2 M Na(+)Cl(-) or K(+)Cl(-) The sensitivity of the simulations to the use of different solvent and ion models is analyzed in detail using multi-microsecond simulations. Finally, an extended (10 μs) simulation is used to characterize slow and infrequent conformational changes in DDD, leading to the identification of previously uncharacterized conformational states of this duplex which can explain biologically relevant conformational transitions. With a total of more than 43 μs of unrestrained molecular dynamics simulation, this study is the most extensive investigation of the dynamics of the most prototypical DNA duplex.
Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.
- MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- fluorescenční spektrometrie MeSH
- FRAP MeSH
- HeLa buňky MeSH
- kinetika MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- prekurzory RNA metabolismus MeSH
- ribonukleoproteiny malé jaderné metabolismus fyziologie MeSH
- sestřih RNA fyziologie MeSH
- spliceozomy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
