Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLF) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E b-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2–5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2–5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC–Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS. Here we show that Bye1 binds with its TFIIS-like domain (TLD) to RNA polymerase (Pol) II, and report crystal structures of the Bye1 TLD bound to Pol II and three different Pol II-nucleic acid complexes. Like TFIIS, Bye1 binds with its TLD to the Pol II jaw and funnel. In contrast to TFIIS, however, it neither alters the conformation nor the in vitro functions of Pol II. In vivo, Bye1 is recruited to chromatin via its TLD and occupies the 5′-region of active genes. A plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4, and this interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and conversely is impaired by repressive PTMs. We identify putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator, which are both implicated in cancer. These results establish Bye1 as the founding member of a unique family of chromatin transcription factors that link histones with active PTMs to transcribing Pol II.gene transcription | chromatin modification F or transcription of eukaryotic protein-coding genes, RNA polymerase (Pol) II associates transiently with dozens of transcription factors. Different Pol II-associated factors are required for transcription initiation, RNA chain elongation through chromatin, pre-mRNA 5′-capping, splicing, 3′-RNA processing of the nascent transcript, and transcription termination (1-3). To understand how these factors cooperate with Pol II and achieve their functions, structural information on Pol II in complex with transcription factors is required. Thus far, X-ray crystallographic structural information on such complexes is limited to two transcription factors: the initiation factor TFIIB (4-7), and the elongation factor TFIIS (8-11). TFIIS contains three domains, a mobile N-terminal domain, a central domain that binds directly to the Pol II jaw and funnel domains, and a C-terminal zinc ribbon domain that inserts into the polymerase pore (also called the secondary channel) and reaches the Pol II active site (9), to stimulate cleavage of backtracked RNA during transcriptional proofreading and escape from arrest (12).In the yeast Saccharomyces cerevisiae, there is only a single protein that contains a domain that is distantly homologous to the central, Pol II-associated domain of TFIIS. This protein, bypass of Ess1 (Bye1), has been identified as a multicopy suppressor of Ess1 (13), a peptidyl-prolyl cis-trans isomerase involved in proline isomerization of the C-terminal domain (CTD) of Pol II (14, 15). In Bye1, the central TFIIS-like domain (TLD, residues 232-365) is flanked by an N-terminal plant homeo domain (PHD) (residues 74-134) and a C-terminal Spen paralogue and orthologue C-terminal (SPOC) domain (residues 447-547; Fig. 1A). PHD domains are mostly found in proteins involved in chromatinmediated gene regulation (16). Con...
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