Research articles

V(D)J recombination is mediated by the products of the recombination activation genes, RAG1 and RAG2. DNA binding and cleavage are targeted by recombination sequences that flank each gene segment and are composed of well-conserved heptamer and nonamer sequences separated either by 12 or 23 base pairs. Schatz and co-workers report the crystal structure of the RAG1 nonamer binding domain (NBD) bound to its cognate sequence. The NBD adopts an intertwined dimer that mediates the synapsis of two DNA molecules. Biochemical and FRET experiments support the structural findings and have implications for the regulation of DNA binding and cleavage by RAG1/2.

Gemin5 is a WD repeat protein that binds small nuclear RNAs (snRNAs) through a specific sequence in the context of the SMN complex, a function required for spliceosomal snRNP biogenesis. Reduced levels of SMN cause spinal muscular atrophy. A series of biochemical experiments now indicate that the WD repeat region of Gemin5 recognizes the snRNAs in a sequence-specific fashion, suggesting that WD repeats are capable of RNA binding.

In eukaryotic cells, DNA is wrapped around histones to form nucleosomes, which are further organized into the 30-nm chromatin fiber. A single-molecule study with homogeneous chromatin fibers now shows that the chromatin fiber behaves as a simple spring, stretching up to three times in response to pulling, a behavior indicative of a one-start helix structure. Linker histones stabilize the fiber but do not make it stiffer.

Production of complex machines such as the ribosome requires coordinated regulation of the components. A widely conserved plant regulator of alternative splicing on the TFIIIA transcription factor mRNA has been found. The RNA structurally mimics the 5S rRNA and, accordingly, binds ribosomal protein L5, which thus affects splicing and production of TFIIIA. As TFIIIA is needed for transcription of the 5S rRNA, this work defines a regulatory circuit for coordinating 5S rRNA production by its binding protein.

A genome-wide analysis of methylated DNA from human embryonic stem cells and adult tissues provides a comprehensive view of unmethylated regions and leads to the identification of sequence motifs that can predict whether a region escapes de novo methylation. This algorithm is used to identify novel, non-CpG unmethylated regions, including intragenic and tissue-specific ones.

The Msh2–Msh3 complex recognizes DNA mismatch lesions, with stronger affinity for small insertion and deletion loops. Now the nucleotide binding properties of Msh2–Msh3 are studied, revealing the changes upon binding to DNA molecules with a loop lesion, indicating how this mismatch sensor can signal the repair machinery.

Most terminally differentiated cells have a diminished capacity to respond to and repair DNA damage. Now a microRNA is shown to have a role in this phenotype in blood cells: miR-24 is upregulated in blood cells differentiated in vitro and decreases the levels of H2AX, a histone variant with a key role in the response to DNA double-stranded breaks.

WAVE proteins in the WASP family are controlled by incorporation into the WAVE regulatory complex (WRC), which transmits information from the Rac GTPase to the actin cytoskeleton. By reconstituting human and fly WRCs, the native complex is shown to be inactive. Rac activates the WRC, but does not cause subunit dissociation. These results reconcile previous work and reveal common regulatory principles for the WASP family.

Dysfunction of SUMO or Rad60 causes overlapping phenotypes that include genomic instability. Now, a nonsubstrate interaction between SUMO-like domain 2 (SLD2) of Rad60 and the SUMO-conjugating enzyme Ubc9 is shown to suppress aberrant replication-associated homologous recombination. Thus, SUMO mimicry provides critical regulation in the SUMO pathway.

Protein synthesis is catalyzed in the peptidyl transferase center of the ribosome. The structure of the 70S ribosome containing tRNAs now gives insight into the active site of a complete ribosome and reveals a direct interaction between the tRNA substrate and ribosomal proteins.

CDC6 is involved in the assembly of pre-replicative complexes. CDC6 is now found to be acetylated by GCN5, a modification that leads to subsequent phosphorylation at sites targeted by by Cyclin A during S phase, thus regulating stability and subcellular localization.

Ribosomal proteins are known to play an important role in determining rRNA structure. The body of the 30S ribosomal subunit is formed by the 16S rRNA 5′ domain. New data indicate that the assembly protein S16 discriminates between folding intermediates of the 5′ domain, increasing cooperative 30S assembly and stabilizing interactions at its decoding site.

The nuclear transport receptor CRM1 mediates protein export from the nucleus through recognition of leucine-rich nuclear export signals on substrates. Structural analysis, based in part on the recent structure of a CRM1-SNUPN complex, reveal determinants for substrate binding and suggest a mechanism for binding partner-assisted dissociation of SNUPN in the cytoplasm.

BMAL1 has a central role in the mammalian circadian clock, acting as a transcriptional activator. The activity of BMAL1 is controlled by post-translational modifications such as acetylation and SUMOylation. Now the kinase CK2a is shown to phosphorylate BMAL1 at Ser90, and this is essential for BMAL1's function in the circadian clock.

MicroRNAs are involved in post-transcriptional regulation of gene expression. Data now indicate that miR-9 targets the nuclear receptor TLX and vice versa, thus proposing a regulatory circuit linked to the switch between neural progenitor proliferation and differentiation.

The conserved A32-U38 pair in the anticodon loop of tRNA^Ala_GGC is now shown to be important for accurate decoding. Using tRNA^Ala variants in this pair in an in vitro translation system showed that some variants could decode both the cognate and a near-cognate codons with high efficiency. These mutants tRNA were toxic in vivo, supporting the biological relevance of this accuracy determinant.

The conserved A32-U38 pair in the anticodon loop of tRNA^Ala_GGC is now shown to be important for accurate decoding. Pre–steady state kinetic analyses of mutants in A32-U38 show that they can efficiently decode near-cognate codons, with a mismatch in any of the three positions, pointing to the role of such conserved sequence elements in suppressing misreading during translation.

Frataxin is a conserved protein involved in the cellular assembly of iron-sulfur clusters, but its exact role is not clear. Now work on bacterial frataxin CyaY indicates that it acts as an iron-dependent regulator of iron-sulfur cluster formation by the desulfurase IscS.

Some viruses, including hepatitis C virus (HCV), bypass cellular initiation factors and can initiate translation through an internal ribosomal entry site (IRES). This process is now examined for the HCV IRES, indicating that conformational changes are necessary but not sufficient for initiation. Instead, the initiator tRNA, but not its interaction with the start codon, seems key to stabilizing HCV mRNA binding to the ribosome, indicating that this IRES bypasses some, but not all, of the functions of the initiation factors.

The La protein binds precursor tRNAs at their 3′ ends, thus facilitating maturation. Crystal structures indicate that the most obvious RNA-recognition motif does not bind this pre-tRNA tail, but studies now indicate that La has two distinct binding sites for the pre-mRNA. Recognition through these two sites may help distinguish precursor from product, an idea supported by biochemical studies here.