ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here, Methanococcus jannaschii MRATPcS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPcS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.

A yeast gene has been identified that encodes a novel, evolutionarily conserved N ␣ -acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A. The gene has been named NAT4. Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin. H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation. In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced. Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group. A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase. Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A. Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.Eukaryotic proteins are subject to two cotranslational modifications as the nascent polypeptides emerge from the ribosome, methionine cleavage by methionine aminopeptidase, and acetylation of the ␣-amino group of the N-terminal amino acid (1). Removal of the methionine occurs only if the second amino acid is a small one such as serine, alanine, or glycine. Acetylation of the ␣-amino group on either methionine, or on the N-terminal residue resulting from methionine cleavage, occurs in the great majority of (but not all) eukaryotic proteins, and is accomplished by one of several N ␣ -acetyltransferases (NATs) 1 (1). A number of years ago we identified the genes for a yeast NAT, now called NatA, that consists of two subunits, Ard1 and Nat1 (2). Subsequent work showed that NatA was responsible for the acetylation of many, but not all, yeast proteins beginning with small residues such as serine, alanine, or glycine (2-5). Yeast ard1 and nat1 mutants are viable and the many proteins that are no longer N-terminally acetylated in the mutant strains are as stable as they are in a wild type strain (2). Thus, there is no evidence that N-terminal acetylation serves to protect proteins from degradation in yeast.Ard1 protein has an acetyl-CoA binding motif found in members of the GNAT superfamily, enzymes that acetylate amino groups on proteins and other molecules (6). Thus, Ard1 is very likely to be the catalytic subunit of NatA. Two other NATs have been identified in yeast, called NatB and NatC, with catalytic subunits Nat3 and Mak3, respectively (4, 7). NatB and NatC both acetylate proteins with N-terminal methionine residues, although they have different specificities dictated by the nature of the subsequent amino acids (4).Histone H4 is a highly conserved protein with an N-terminal serin...

Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169-293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329-530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).

The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3′-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated β-actin or eEF2 mRNA to eIF4E-bound polyadenylated β-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3′-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs.

Background: How the eIF3 complex and ribosomes are recruited during translation on CBP80/20-bound mRNAs remains obscure. Results: CTIF interacts with eIF3g to recruit the eIF3 complex. Conclusion: Translation on CBP80/20-bound mRNAs requires CTIF-eIF3g interaction. Significance: The use of different eIF3 subunits for recruiting eIF3 complex implies that translation on CBP80/20-bound mRNAs differs mechanistically from translation on eIF4E-bound mRNAs.