A novel occluded RNA recognition motif in Prp24 unwinds the U6 RNA internal stem loop

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

The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors

In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3′- and 5′-ends of 261 yeast genes by run-on. The results obtained indicate that the 3′/5′ run-on ratio varies among the genes studied by over 12 log2 units. This ratio seems to be an intrinsic characteristic of each transcriptional unit and does not significantly correlate with gene length, G + C content or level of expression. The correlation between the 3′/5′ RNA polymerase II ratios measured by run-on and those obtained by chromatin immunoprecipitation is poor, although the genes encoding ribosomal proteins present exceptionally low ratios in both cases. We detected a subset of elongation-related factors that are important for maintaining the wild-type profiles of active transcription, including DSIF, Mediator, factors related to the methylation of histone H3-lysine 4, the Bur CDK and the RNA polymerase II subunit Rpb9. We conducted a more detailed investigation of the alterations caused by rpb9Δ to find that Rpb9 contributes to the intragenic profiles of active transcription by influencing the probability of arrest of RNA polymerase II

Étude systématique des domaines protéiques dans le cadre d'un projet de génomique structurale (application au cas de la protéine de levure Set1)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Structural Basis for Phosphoinositide Substrate Recognition, Catalysis, and Membrane Interactions in Human Inositol Polyphosphate 5 Phosphatases

SummarySHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family

Structural Basis for Potency and Promiscuity in Poly ADP ribose Polymerase PARP and Tankyrase Inhibitors

Selective inhibitors could help unveil the mechanisms by which inhibition of poly­(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects

Substrate Specificity and Oligomerization of Human GMP Synthetase

AbstractGuanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors

An automatable screen for the rapid identification of proteins amenable to refolding

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under nondenaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism