No abstract

The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.

Translational initiation factor 2 (IF2) is a guanine nucleotidebinding protein that can bind guanosine 3 ,5 -(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.fast kinetics ͉ GTP ͉ translation regulation ͉ nutritional stress I nitiation factor 2 (IF2) is the only initiation factor that is ribosome-bound throughout the entire translation initiation pathway, participating initially in the formation of the 30S initiation complex (30SIC) and subsequently in the assembly of the 70S initiation complex (70SIC), a process that ultimately results in formation of the first peptide bond (initiation dipeptide) and generates the first ribosomal pretranslocation complex (for reviews, see refs. 1-5). Thus, it could be predicted that IF2 functions are accompanied͞modulated by conformational changes that could be either consequence or cause of the interactions of IF2 with its various ligands (30S and 50S ribosomal subunits, fMet-tRNA, GTP, GDP⅐Pi, and GDP). Crystallographic (6) and NMR (7) studies have, in fact, shown that, depending upon the nature of their ligand (i.e., GTP or GDP), aIF5B, the archaeal homologue of bacterial IF2, as well as isolated IF2G2, the G domain of IF2, undergo large structural changes. On the other hand, chemical probing (8) and cryo-EM (9, 10) have clearly shown that several conformational changes of the factor occur during the early, middle, and late events of translation initiation.Bacterial cells growing under optimal nutritional conditions contain a high (Ͼ1 mM) concentration of GTP and a vanishingly low level of GDP. Thus, IF2 is expected to exist and to bind the 30S subunit almost exclusively in the GTP form, because it displays similar affinity for GTP and GDP, both K d s being in the 10-to 100-M range (11). The IF2⅐GTP was shown to have a higher affinity for the 30S ribosomal subunit than either IF2⅐GDP or free IF2 (11). The adjustment of fMet-tRNA in the P site (ref. 12 and refs. therein) and the release of IF2 from 70SIC (13-15) have been attributed to the IF2-dependent GTP hydrolysis, which is very rapidly triggered by th...

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization-based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.