The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat.

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS. DOI:http://dx.doi.org/10.7554/eLife.00327.001

Structural studies of proteins from the recfor pathway involved in dna repair by homologous recombination.

Maintenance of genomic integrity is extremely important for all organisms. Thus, all cells are equipped with DNA repair mechanisms for different types of DNA damage. The function of the recFOR pathway of recombination is currently not well understood. In E.coli, the recFOR pathway contributes only 0.1-1% of the recombinational activity in the cell (Horii and Clark, 1973) and recF and recR mutants have relatively subtle phenotypes with regard to recombination. Their UV sensitivity is, however, greatly increased. Studies by Courcelle et al. (1997) suggested that recF and recR are required for the resumption of replication at stalled DNA replication forks. Hence, the primary function of proteins in the recFOR pathway in E.coli may not be recombination, but resumption of DNA replication from existing replication forks. In order to get a better understanding of the structure and function of the proteins constituting the RecFOR pathway, we are have purified RecF, RecO, and RecR, and crystallized several of them. The goal is to determine the structure of the proteins alone and/or in complex with each other and with DNA. Currently, diffraction data of RecO crystals with and without bound oligonucleotide have been collected to 2.5 ? and 2.8 ? respectively. Crystals of RecR have been grown but need further improvement. Biochemical experiments provide functional data to determine the partners in complex formation and DNA binding activity of the various complexes. Horii, Z. and Clark, A.J. (1973). J. Mol. Biol. 80, 327-344.Courcelle, J., Carswell-Crumpton, C., Hanawalt, P.C. (1997). Proc. Natl. Acad. Sci. USA 94: 3714-3719

Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex.

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM

Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide

RecFOR proteins are important for DNA repair by homologous recombination in bacteria. The RecO protein from Thermus thermophilus was cloned, purified and characterized for its binding to oligonucleotides. The protein was crystallized alone and in complex with a 14-mer oligonucleotide. Both crystal forms grow under different crystallization conditions in the same space group, P3121 or P3221, with almost identical unit cell parameters. Complete data sets were collected to 2.8 Angstrom and 2.5 Angstrom for RecO alone and the RecO-oligonucleotide complex, respectively. Visual comparison of the diffraction patterns between the two crystal forms and calculation of an Rmerge of 33.9 percent on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide

TOWARDS CONFORMATIONAL SEQUENCING OF PROTEINS: ASSIGNMENT OF SECONDARY STRUCTURES BY ANTI-PEPTIDE ANTIBODIES

We performed model studies towards assignment of &-turn and a-helices to protein primary structures with antibodies. Probing of a &-turn was attempted with anti-peptide antibodies directed against the &-turn (DPGQ) of a synthetic &-turn model-peptide (IVIVIDPGQTVTY) adopting the intended conformation &-sheet-&-turn-&-sheet. The specific anti-&-turn model-peptide antibodies have a three orders of magnitude higher affinity for the &-turn containing epitope than the control Gly-peptide (GsDPGQG,, ) of random coil structure. The antibody affinity for the &-turn region (DPGQ) increases from the primary to the hyperimmune response. Although the chosen &-turn sequence is similar to parts of the animal's own proteins, self-tolerance did not raise difficulties in generating antibodies against the R-turn model-peptide. Individual putative &-turn sequences of proteins may be probed by including their sequence between the two &-sheet cartridges of the &-turn model-peptide. Helix assignment was probed with synthetic model peptides of extended conformation including only the superimposed residues of a putative helix (every fourth residue) linked by a spacer amino acid residue (alanine throughout or the corresponding third residue of the sequence to be tested) in order to adjust the translation of the relevant residues of the model-peptide to the helical pitch. The anti-helix modelpeptide antibodies were shown by Western blotting to react in a sequence-specific manner with the corresponding model protein lactose permease of E.coli. "Conformational sequencing" i.e. sequence assignments of secondary structures by anti-peptide antibodies now seems feasable for &-turn regions and helices of proteins of known sequence