Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoylphenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiledcoil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.
Background: Yeast Sen1 helicase and its human ortholog senataxin promote accurate transcription termination. Results: Sen1 helicase domain exhibits 5Ј-to 3Ј-helicase activity on DNA and RNA and binds endogenous RNA. Conclusion: Biochemical activities of purified Sen1 helicase domain are consistent with its proposed function in resolving cotranscriptional R-loops. Significance: Mutations in senataxin and its paralog IGHMBP2 cause crippling neurodegenerative diseases.
The essential splicing factor Prp24 contains four RNA Recognition Motif (RRM) domains, and functions to anneal U6 and U4 RNAs during spliceosome assembly. Here, we report the structure and characterization of the C-terminal RRM4. This domain adopts a novel non-canonical RRM fold with two additional flanking α-helices that occlude its β-sheet face, forming an occluded RRM (oRRM) domain. The flanking helices form a large electropositive surface. oRRM4 binds to and unwinds the U6 internal stem loop (U6 ISL), a stable helix that must be unwound during U4/U6 assembly. NMR data indicate that the process starts with the terminal base pairs of the helix and proceeds toward the loop. We propose a mechanistic and structural model of Prp24′s annealing activity in which oRRM4 functions to destabilize the U6 ISL during U4/U6 assembly.
U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24's four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (59-GAGA-39), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24's annealing activity is proposed.
Saccharomyces cerevisiae Prp24 is an essential RNA binding protein involved in pre-mRNA splicing. Nearly complete backbone and side chain resonance assignments have been obtained for the second RNA recognition motif (RRM) of Prp24 (RRM2, residues M114-E197) both in isolation and bound to a 6 nucleotide fragment of U6 RNA (AGAGAU). In addition, nearly complete backbone assignments have been made for a Prp24 construct spanning the second and third RRMs (RRM23, residues M114-K290), both free and bound to AGAGAU. KeywordsRRM; protein-RNA complex; splicing; snRNP Biological ContextProper eukaryotic gene expression requires successful pre-mRNA splicing, catalyzed by the spliceosome . The spliceosome is assembled from five RNA-protein complexes (small nuclear ribonucleoproteins, or snRNPs). Each snRNP contains one small nuclear RNA molecule (named U1, U2, U4, U5, or U6) and multiple proteins. The Saccharomyces cerevisiae protein Prp24 is required for incorporation of U6 RNA into the spliceosome (Shannon and Guthrie 1991;Raghunathan and Guthrie 1998). Prp24 binds to U6 RNA as a part of the U6 snRNP (Karaduman et al. 2006;Karaduman et al. 2008), and facilitates basepairing between U6 RNA and U4 RNA (Shannon and Guthrie 1991;Ghetti et al. 1995).Prp24 contains four RNA recognition motif (RRM) domains (Bae et al. 2007). Previous work established that RRM1 and/or RRM2 of Prp24 bind the AGAGAU sequence of U6 RNA (Kwan and Brow 2005;Bae et al. 2007). Interestingly, the canonical RNA binding surface of RRM2 appears to be occluded by inter-domain contacts in a crystal structure (Bae et al. 2007). In order to further investigate RNA binding by RRM2 of Prp24, and the inter-domain orientation between RRM2 and RRM3, we have obtained backbone and side chain assignments for RRM2 free and bound to AGAGAU RNA, and backbone assignments for RRM23 free and bound to AGAGAU. Ethics and Conflicts:The authors declare that they have no conflict of interest, and that the experiments performed comply with the current laws of the United States of America. NIH Public Access Author ManuscriptBiomol NMR Assign. Author manuscript; available in PMC 2010 November 3. Methods and ExperimentsUniformly unlabeled, 15 N, and 13 C 15 N labeled protein (RRM2-His 6 or RRM23-His 6 ) was prepared through over-expression from a pET21b plasmid transformed into BL21 E. coli (Stratagene). Cells were grown to saturation in 750 mL M9 minimal media supplemented with 13 C glucose and/or 15 N ammonium chloride as needed. Unlabeled RRM2 was grown in Luria Broth. Protein production was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 15 hours at room temperature. Cells were pelleted by centrifugation and stored at −80°C. Frozen cell pellets were thawed for 15 hours at 4°C, and then resuspended in 50 mL Wash Buffer (50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 10 mM imidazole). Lysis was performed on the 50 mL volume using a 30 minute incubation with 1 mg/mL lysozyme at 4°C, followed by 2 minutes sonication on ice (12 cycles of a 10 second...
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