English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

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

MPS-Authors
/persons/resource/persons32560

Ohrt,  T.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15595

Odenwälder,  P.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons14960

Dannenberg,  J.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15993

Warkocki,  Z.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15777

Schmitzova,  J.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15301

Karaduman,  R.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15048

Fabrizio,  P.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

/persons/resource/persons15470

Lührmann,  R.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

1818584.pdf
(Publisher version), 498KB

Supplementary Material (public)

1818584-Suppl-1.pdf
(Supplementary material), 3MB

Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-FD14-3
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.