Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments.

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5' UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, "humanized" yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.

Structural basis and mechanism for metallochaperone-assisted assembly of the CuA center in cytochrome oxidase.

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+•Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu2+•CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC•Cu1+•Cu2+ can release CoxB•Cu2+ from the ScoI•Cu2+•CoxB complex. The CoxB•CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC•Cu1+•Cu2+ to CoxB•Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

The use of ene adducts to study and engineer enoyl-thioester reductases.

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.

How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

Purification, crystallization and preliminary X-ray analysis of the effector domain of AlsR, an LysR-type transcriptional regulator from Bacillus subtilis.

AlsR from Bacillus subtilis, a member of the LysR-type transcriptional regulator (LTTR) family, regulates the transcription of the alsSD operon encoding enzymes involved in acetoin biosynthesis. LTTRs represent the largest known family of transcriptional regulators in bacteria. In this study, AlsR82-302S100A, representing the effector domain, was produced in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method in the presence of 2.1 M DL-malic acid pH 7.0 at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 142.91, b = 74.96, c = 94.39 Å, ß = 110.543°. X-ray data extending to a resolution of 2.6 Šwere collected.

Structural basis for intrinsic thermosensing by the master virulence regulator RovA of Yersinia.

Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment.

Unusual carbon fixation gives rise to diverse polyketide extender units.

Polyketides are structurally diverse and medically important natural products that have various biological activities. During biosynthesis, chain elongation uses activated dicarboxylic acid building blocks, and their availability therefore limits side chain variation in polyketides. Recently, the crotonyl-CoA carboxylase-reductase (CCR) class of enzymes was identified in primary metabolism and was found to be involved in extender-unit biosynthesis of polyketides. These enzymes are, in theory, capable of forming dicarboxylic acids that show any side chain from the respective unsaturated fatty acid precursor. To our knowledge, we here report the first crystal structure of a CCR, the hexylmalonyl-CoA synthase from Streptomyces sp. JS360, in complex with its substrate. Structural analysis and biochemical characterization of the enzyme, including active site mutations, are reported. Our analysis reveals how primary metabolic CCRs can evolve to produce various dicarboxylic acid building blocks, setting the stage to use CCRs for the production of unique extender units and, consequently, altered polyketides.

Structure of the effector-binding domain of the LysR-type transcription factor RovM from Yersinia pseudotuberculosis.

In enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 Å. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.

Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is responsible for around 10% of all hospital-acquired infections and the single most important pathogen of cystic fibrosis lungs. P. aeruginosa has high intrinsic and acquired antibiotic resistance, due to the extrusion of antibiotics by multidrug efflux pumps. The gene regulator MexZ controls the expression of mexXY, the efflux pump responsible for resistance to many drugs that are used for treating CF patients. MexZ is shown to be the most frequently mutated gene in P. aeruginosa isolated from CF patient lungs, confirming its importance in multidrug resistance. Here we present the crystal structure of MexZ at 2.9Å. Combining the structural information with biochemical data on key mutants identified, we provide an explanation for the structural and functional consequences of these mutants. This work provides a framework for further characterisation of MexZ in order to fully understand its regulation and induction.