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  Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

Ohrt, T., Odenwälder, P., Dannenberg, J., Prior, M., Warkocki, Z., Schmitzova, J., et al. (2013). Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system. RNA, 19(7), 902-915. doi:10.1261/rna.039024.113.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0013-FD14-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-8231-C
Genre: Journal Article

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 Creators:
Ohrt, T.1, Author              
Odenwälder, P.1, Author              
Dannenberg, J.1, Author              
Prior , M., Author
Warkocki, Z.1, Author              
Schmitzova, J.1, Author              
Karaduman, R.1, Author              
Gregor, I., Author
Enderlein, J., Author
Fabrizio, P.1, Author              
Lührmann, R.1, Author              
Affiliations:
1Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578576              

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Free keywords: spliceosome; step 2 catalysis; step 2 factors/Prp16; Slu7 Prp18; Prp22
 Abstract: Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3' SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3' SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

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Language(s): eng - English
 Dates: 2013-05-172013-07
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1261/rna.039024.113
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Title: RNA
Source Genre: Journal
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Pages: - Volume / Issue: 19 (7) Sequence Number: - Start / End Page: 902 - 915 Identifier: ISSN: 1355-8382