Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of ϳ130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys 48 linkages, having little or no activity on Lys 63 -and Lys 29 -linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA’s cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.

Background:The survival motor neuron (SMN) protein forms oligomeric complexes involved in ribonucleoprotein (RNP) biogenesis. Results: SMN forms stable dimers, which in turn self-associate to form tetramers and octamers. Conclusion: SMN complexes form discrete oligomers with unusually large hydrodynamic sizes. Significance: Understanding the oligomeric nature of SMN provides an important foundation for exploring the biochemical bases of RNP assembly and spinal muscular atrophy.

The posttranscriptional modifications of tRNA's anticodon stem and loop (ASL) domain represent a third level, a third code, to the accuracy and efficiency of translating mRNA codons into the correct amino acid sequence of proteins. Modifications of tRNA's ASL domain are enzymatically synthesized and site specifically located at the anticodon wobble position-34 and 3'-adjacent to the anticodon at position-37. Degeneracy of the 64 Universal Genetic Codes and the limitation in the number of tRNA species require some tRNAs to decode more than one codon. The specific modification chemistries and their impact on the tRNA's ASL structure and dynamics enable one tRNA to decode cognate and "wobble codons" or to expand recognition to synonymous codons, all the while maintaining the translational reading frame. Some modified nucleosides' chemistries prestructure tRNA to read the two codons of a specific amino acid that shares a twofold degenerate codon box, and other chemistries allow a different tRNA to respond to all four codons of a fourfold degenerate codon box. Thus, tRNA ASL modifications are critical and mutations in genes for the modification enzymes and tRNA, the consequences of which is a lack of modification, lead to mistranslation and human disease. By optimizing tRNA anticodon chemistries, structure, and dynamics in all organisms, modifications ensure translational fidelity of mRNA transcripts.

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN–Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure–function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.

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Small‐angle neutron scattering (SANS) with contrast variation can provide useful information about the structure and disposition of two or more chemically distinct components within a complex. The SASSIE Contrast Calculator (SCC) is a new software tool designed to assist in planning SANS experiments with contrast variation on protein and nucleic acid complexes. On the basis of the primary sequence and deuteration level of each protein or nucleic acid component, the SCC calculates and plots I(0), contrast and scattering length densities; since SANS experiments often complement small‐angle X‐ray scattering studies, the program provides both neutron and X‐ray parameters. The SCC is run as an integrated component of SASSIE [Curtis, Raghunandan, Nanda & Krueger (2012). Comput. Phys. Commun.183, 382–389], a software suite for atomistic modeling of ensembles of structures consistent with scattering data.

Background: Threonylcarbamoyl-AMP synthase catalyzes formation of the biosynthetic intermediate of a critical tRNA modification, t 6 A 37 . Results: Structural analyses provide insight into the interaction between the substrates ATP and L-threonine and the E. coli enzyme. Conclusion:The threonylcarbamoyl-AMP synthase binds L-threonine and ATP cooperatively; L-threonine is required for positioning of ATP. Significance: Mechanistic insights into t 6 A 37 biosynthesis provide an understanding of this complex enzymatic pathway.