Expanding the structural repertoire of g-quadruplexes

A crystal structure in this issue by Pan and colleagues adds a new dimension to nucleic acid quadruplex structural biology by revealing an RNA quadruplex with bulged uridines.     

Protein-derived cofactors. Expanding the scope of post-translational modifications

Recent advances in enzymology, structural biology, and protein chemistry have extended the scope of the field of cofactor-dependent enzyme catalysis. It has been documented that catalytic and redox-active prosthetic groups may be derived from post-translational modification of amino acid residues of proteins. These protein-derived cofactors typically arise from the oxygenation of aromatic residues, covalent cross-linking of amino acid residues, or cyclization or cleavage of internal amino acid residues. In some cases, the post-translation modification is a self-processing event, whereas in others, another processing enzyme is required. The characterization of protein-derived cofactors and their mechanisms of biogenesis introduce a new dimension to our current views about protein evolution and protein structure-function relationships.     

CryoEM structures of pseudouridine-free ribosome suggest impacts of chemical modifications on ribosome conformations

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Expanding the repertoire of optogenetically targeted cells with an enhanced gene expression system

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.     

Expanding the morphogenetic repertoire: perspectives from the Drosophila egg

Tissue and organ architectures are incredibly diverse, yet our knowledge of the morphogenetic behaviors that generate them is relatively limited. Recent studies have revealed unexpected mechanisms that drive axis elongation in the Drosophila egg, including an unconventional planar polarity signaling pathway, a distinctive type of morphogenetic movement termed "global tissue rotation," a molecular corset-like role of extracellular matrix, and oscillating basal cellular contractions. We review here what is known about Drosophila egg elongation, compare it to other instances of morphogenesis, and highlight several issues of general developmental relevance.     

rRNA modifications in an intersubunit bridge of the ribosome strongly affect both ribosome biogenesis and activity

The presence of nucleotide modifications in rRNA has been known for nearly 40 years; however, information about their roles is sparse. Here, we describe the consequences of depleting modifications from an intersubunit bridge (helix 69) of the ribosomal large subunit in yeast. Helix 69 interacts with both A and P site tRNAs and contains five modifications. Blocking one to two modifications has no apparent effect on cell growth, whereas loss of three to five modifications impairs growth and causes the broadest defects observed thus far for modification loss in any ribosome region. Major effects include the following: (1) reduced amino acid incorporation rates in vivo (25%-60%); (2) increased stop codon readthrough activity; (3) increased sensitivity to ribosome-based antibiotics; (4) reduced rRNA levels (20%-50%), due mainly to faster turnover; and (5) altered rRNA structure in the ribosome. Taken together, the results indicate that this subset of rRNA modifications can influence both ribosome synthesis and function and in synergistic ways.     

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Expanding the repertoire of nitrilases with broad substrate specificity and high substrate tolerance for biocatalytic applications

Enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. Although nitrilases from several sources have been characterized, there exists a scope for identifying broad spectrum nitrilases exhibiting higher substrate tolerance and better thermostability to develop industrially relevant biocatalytic processes. Through genome mining, we have identified nine novel nitrilase sequences from bacteria and evaluated their activity on a broad spectrum of 23 industrially relevant nitrile substrates. Nitrilases from Zobellia galactanivorans, Achromobacter insolitus and Cupriavidus necator were highly active on varying classes of nitriles and applied as whole cell biocatalysts in lab scale processes. Z. galactanivorans nitrilase could convert 4-cyanopyridine to achieve yields of 1.79 M isonicotinic acid within 3 h via fed-batch substrate addition. The nitrilase from A. insolitus could hydrolyze 630 mM iminodiacetonitrile at a fast rate, effecting 86 % conversion to iminodiacetic acid within 1 h. The arylaliphatic nitrilase from C. necator catalysed enantioselective hydrolysis of 740 mM mandelonitrile to (R)-mandelic acid in 4 h. Significantly high product yields suggest that these enzymes would be promising additions to the suite of nitrilases for upscale biocatalytic application.     

Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers

Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications. The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure. Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites. Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites. The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker. BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322. BsmI excision linkers are also described.     

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.