Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role

(1)H, (13)C and (15)N assignments of CdnL, an essential protein in Myxococcus xanthus

CdnL, an essential protein in Myxococcus xanthus and several other bacteria, is a member of the large CarD_TRCF family of bacterial proteins that interact with RNA polymerase. Structural analyses of the 164-residue M. xanthus CdnL by NMR is complicated because of broadening, and hence overlap, of the signals due to the self-association and the monomer–dimer equilibrium that occurs in solution. Here, we report 1H, 13C and 15N assignments for CdnL achieved by analyzing its NMR spectra on the basis of the complete assignment obtained in this study for the 68-residue N-terminal fragment of CdnL (CdnLNt) together with those we described previously for the stable, protease-resistant, 110-residue C-terminal domain (CdnLCt). This approach relied on our observation that many of the CdnLNt and CdnLCt chemical shifts matched closely with those of the equivalent residues in the full-length protein. Our assignments provide the crucial first step in the structural analysis of CdnL and this functionally important family of bacterial proteins.This work was funded by grants CTQ2008- 0080/BQU (MAJ), BFU2009-12445-C02-02 (SP) and BFU2009- 12445-C02-01 (MEA) from the Ministerio de Ciencia e Innovación (MCINN), Spain, and a Ph.D. fellowship (YM) from Consejo Superior de Investigaciones Científicas (CSIC), Spain.Peer Reviewe

Structural insights into mrnp formatlon

Resumen del póster presentado en la IV Reunión bienal del grupo de especialización de RMN de la RSEQ. I Reunión Ibérica de RMN (2008), celebrada en Sevilla (España) del 21 al 24 de septiembre de 2008

NMR structure note: N-terminal domain of Thermus thermophilus CdnL

9 pags, 3 figs, 1 tabThis work was supported by the Ministerio de Ciencia e Innovación, Spain grants: CTQ2008-0080/BQU and CTQ2011-22514 (MAJ), BFU2009-12445-C02-02 (SP), BFU2009-12445-C02-01(MEA) co-financed by the European Union (FEDER) and Ph.D. fellowships from the Ministerio de Ciencia e Innovación, Spain (AGG) and the Consejo Superior de Investigaciones Científicas, Spain (YM)

Two singular types of CCCH tandem zinc finger in Nab2p contribute to polyadenosine RNA recognition

12 pags, 7 figs, 2 tabs. -- Supplemental Information is available at the Publisher´s webThe seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 μM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains. © 2013 Elsevier Ltd.This work was supported by a CSIC Special Intramural project (200880I088), a MINECO project (CTQ2011-26665), and a “Comunidad Autónoma de Madrid” project (CCG07-CSIC GEN2333). S.M.L. holds an undergraduate contract from the “Consejeria de Educacion de la Comunidad Autónoma de Madrid” and the European Social Fund

Disulfide bonds versus Trp···Trp pairs in irregular β-hairpins: NMR structure of vammin loop 3-derived peptides as a case study

9 pages, 6 figures, 1 table.-- PMID: 19294654 [PubMed].-- Supporting information (Suppl. figs SF1-SF2, tabs ST1-ST2) available at: http://www3.interscience.wiley.com/cgi-bin/fulltext/122264837/sm001.pdf?PLACEBO=IE.pdfWhere a noncovalent interaction is better than a covalent bond: The most stabilising cross-strand pairs were incorporated into an irregular β-hairpin, loop 3 of vammin. 1H and 13C NMR conformational analyses of these designed peptides indicated that an edge-to-face Trp···Trp interaction leads to a β-hairpin that is more stable than a disulfide bond.Structural studies on model peptides have led to a good understanding of the rules behind the formation and stability of regular β-hairpins. To test their applicability to the successful design of irregular β-hairpins with long loops and/or β-bulges at the strands, we mimicked loop 3 of vammin, a 4:6 β-hairpin with a non-Gly β-bulge. The most stabilising cross-strand pairs, disulfide bonds or/and Trp···Trp pairs, were incorporated at non-hydrogen-bonded sites in peptides spanning the 69-80 region of vammin. According to NMR data, these modified peptides adopt β-hairpin conformations as intended by design. The Trp-containing peptides reproduce even the unusual positive φ angle for the Gln residue, with the indole rings in the preferred edge-to-face orientation. For the first time the β-hairpin-stabilising capacities of a disulfide bond and a Trp···Trp pair are compared in the same model system. We found that the contribution to stability of the noncovalent indole-indole interaction is larger than that of the covalent disulfide bond, and that their combination gives rise to an even more stable β-hairpin.We thank financial support from CSIC Intramural projects 200580F0161 and 200580F0162 and from MICINN projects CTQ2008-00080/BQU and SAF2006-01205. C.M.S. acknowledges the CSIC I3P program for financial support from the European Social Fund.Peer reviewe

Structural insights into RNA polymerase recognition and essential function of Myxococcus xanthus CdnL

© 2014 Gallego-García et al. CdnL and CarD are two functionally distinct members of the CarD-CdnL-TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in Myxococcus xanthus. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) s-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in M. xanthus. Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded β-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-β, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σA-RNAP holoenzyme at an rRNA promoter in vitro. Consistent with this, CdnL is present at rRNA promoters in vivo. Implication of CdnL in RNAP-σA activity and of CarD in ECF-σ function in M. xanthus exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.Peer Reviewe

Structural Insights into RNA Polymerase Recognition and Essential Function of <i>Myxococcus xanthus</i> CdnL

<div><p>CdnL and CarD are two functionally distinct members of the CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in <i>Myxococcus xanthus</i>. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) σ-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in <i>M. xanthus.</i> Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded β-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-β, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σ^A-RNAP holoenzyme at an rRNA promoter <i>in</i><i>vitro</i>. Consistent with this, CdnL is present at rRNA promoters <i>in</i><i>vivo.</i> Implication of CdnL in RNAP-σ^A activity and of CarD in ECF-σ function in <i>M. xanthus</i> exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.</p></div