Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154520/1/fsb2009014006.pd

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins

The Nucleolus: A Multiphase Condensate Balancing Ribosome Synthesis and Translational Capacity in Health, Aging and Ribosomopathies

The nucleolus is the largest membrane-less structure in the eukaryotic nucleus. It is involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and is the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the intricate pathophysiological relationship between the nucleolus and protein synthesis has only recently begun to emerge. Here, we provide perspective on new principles governing nucleolar formation and the resulting multiphase organization driven by liquid-liquid phase separation. With recent advances in the structural analysis of ribosome formation, we highlight the current understanding of the step-wise assembly of pre-ribosomal subunits and the quality control required for proper function. Finally, we address how aging affects ribosome genesis and how genetic defects in ribosome formation cause ribosomopathies, complex diseases with a predisposition to cancer

Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins

α-Sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S–28S rRNA. α-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast k cat/Km ∼109–1010 M−1s−1, implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity

Metals, Motifs, and Recognition in the Crystal Structure of a 5S rRNA Domain

AbstractTwo new RNA structures portray how non-Watson-Crick base pairs and metal ions can produce a unique RNA shape suitable for recognition by proteins. The crystal structures of a 62 nt domain of E. coli 5S ribosomal RNA and a duplex dodecamer encompassing an internal loop E have been determined at 3.0 and 1.5 Å, respectively. This loop E region is distorted by three “cross-strand purine stacks” and three novel, water-mediated noncanonical base pairs and stabilized by a four metal ion zipper. These features give its minor groove a unique hydrogen-bonding surface and make the adjacent major groove wide enough to permit recognition by the ribosomal protein L25, which is expected to bind to this surface

The common and the distinctive features of the bulged-G motif based on a 1.04 Å resolution RNA structure

Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs. To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures. The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs). The wild-type SRL has now been determined at 1.04 Å resolution, supplementing data obtained before at 1.11 Å and allowing the first calculation of coordinate error for an RNA motif. The other two structures, having a viable (C2658U(•)G2663A) or a lethal mutation (C2658G(•) G2663C), were determined at 1.75 and 2.25 Å resolution, respectively. Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements. Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves. Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G(•)G2663C mutation and may underlie the lethal phenotype

Comparison of the crystal and solution structures of two RNA oligonucleotides.

Until recently, there were no examples of RNAs whose structures had been determined by both NMR and x-ray crystallography, and thus there was no experimental basis for assessing the accuracy of RNA solution structures. A comparison of the solution and the crystal structures of two RNAs is presented, which demonstrates that NMR can produce solution structures that resemble crystal structures and thus validates the application to RNA of a methodology developed initially for the determination of protein conformations. Models for RNA solution structures are appreciably affected by the parameters used for their refinement that describe intramolecular interactions. For the RNAs of interest here, the more realistic those parameters, the greater the similarity between solution structures and crystal structures