Functional role and ribosomal position of the unique N-terminal region of DHX29, a factor required for initiation on structured mammalian mRNAs.

Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377-448) whereas linker corresponds to the long α-helix (aa 460-512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90-166) that constitutes a previously unassigned section of the main density and resides between DHX29's C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29's function.

Structure of mammalian eIF3 in the context of the 43S preinitiation complex.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Šresolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit.

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29.

Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAi(Met) in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs.

On most eukaryotic mRNAs, initiation codon selection involves base-by-base inspection of 5' UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa-long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29's activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNA-binding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29's function in initiation and validated the significance of a ß-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29's intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.

A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae.

The 5'-untranslated regions (5' UTRs) of picornavirus genomes contain an internal ribosomal entry site (IRES) that promotes the end-independent initiation of translation. Picornavirus IRESs are classified into four structurally distinct groups, each with different initiation factor requirements. Here, we identify a fifth IRES class in members of Kobuvirus, Salivirus, and Paraturdivirus genera of Picornaviridae: Aichi virus (AV), bovine kobuvirus (BKV), canine kobuvirus (CKoV), mouse kobuvirus (MKoV), sheep kobuvirus (SKV), salivirus A (SV-A), turdivirus 2 (TV2), and TV3. The 410-nucleotide (nt)-long AV IRES comprises four domains (I to L), including a hairpin (L) that overlaps a Yn-Xm-AUG (pyrimidine tract/spacer/initiation codon) motif. SV-A, CKoV, and MKoV also contain these four domains, whereas BKV, SKV, and TV2/TV3 5' UTRs contain domains that are related to domain I and equivalent to domains J and K but lack an AV-like domain L. These IRESs are located at different relative positions between a conserved 5'-terminal origin of replication and divergent coding sequences. Elements in these IRESs also occur elsewhere: domain J's apical subdomain, which contains a GNRA tetraloop, matches an element in type 1 IRESs, and eIF4G-binding motifs in domain K and in type 2 IRESs are identical. Other elements are unique, and their presence leads to unique initiation factor requirements. In vitro reconstitution experiments showed that like AV, but in contrast to other currently characterized IRESs, SV-A requires the DExH-box protein DHX29 during initiation, which likely ensures that the initiation codon sequestered in domain L is properly accommodated in the ribosomal mRNA-binding cleft.

Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling.

Eukaryotic translation initiation begins with ribosomal recruitment of aminoacylated initiator tRNA (Met-tRNA(Met)(i)) by eukaryotic initiation factor eIF2. In cooperation with eIF3, eIF1, and eIF1A, Met-tRNA(Met)(i)/eIF2/GTP binds to 40S subunits yielding 43S preinitiation complexes that attach to the 5'-terminal region of mRNAs and then scan to the initiation codon to form 48S initiation complexes with established codon-anticodon base-pairing. Stress-activated phosphorylation of eIF2alpha reduces the level of active eIF2, globally inhibiting translation. However, translation of several viral mRNAs, including Sindbis virus (SV) 26S mRNA and mRNAs containing hepatitis C virus (HCV)-like IRESs, is wholly or partially resistant to inhibition by eIF2 phosphorylation, despite requiring Met-tRNA(Met)(i). Here we report the identification of related proteins that individually (Ligatin) or together (the oncogene MCT-1 and DENR, which are homologous to N-terminal and C-terminal regions of Ligatin, respectively) promote efficient eIF2-independent recruitment of Met-tRNA(Met)(i) to 40S/mRNA complexes, if attachment of 40S subunits to the mRNA places the initiation codon directly in the P site, as on HCV-like IRESs and, as we show here, SV 26S mRNA. In addition to their role in initiation, Ligatin and MCT-1/DENR can promote release of deacylated tRNA and mRNA from recycled 40S subunits after ABCE1-mediated dissociation of post-termination ribosomes.

Biosynthesis of the aminocyclitol subunit of hygromycin A in Streptomyces hygroscopicus NRRL 2388.

The antibacterial activity of hygromycin A (HA) arises from protein synthesis inhibition and is dependent upon a methylenedioxy bridged-aminocyclitol moiety. Selective gene deletions and chemical complementation in Streptomyces hygroscopicus NRRL 2388 showed that the hyg18 and hyg25 gene products, proposed to generate a myo-inositol intermediate, are dispensable for HA biosynthesis but contribute to antibiotic yields. Hyg8 and Hyg17, proposed to introduce the amine functionality, are essential for HA biosynthesis. Hyg6 is a methyltransferase acting on the aminocyclitol, and a Deltahyg6 mutant produces desmethylenehygromycin A. Deletion of hyg7, a metallo-dependant hydrolase homolog gene, resulted in methoxyhygromycin A production, demonstrating that the corresponding gene product is responsible for the proposed oxidative cyclization step of methylenedioxy bridge formation. The methyl/methylene group is not required for in vitro protein synthesis inhibition but is essential for activity against Escherichia coli.

The final step of hygromycin A biosynthesis, oxidation of C-5''-dihydrohygromycin A, is linked to a putative proton gradient-dependent efflux.

Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5''-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in Escherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the Deltahyg19 or the Deltahyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.

An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus.

The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (K(m) = 30 +/- 4 microM) at the C-2''' position of the fucofuranose ring in the presence of ATP (K(m) = 200 +/- 20 microM) or GTP (K(m) = 350 +/- 60 microM) with a k(cat) of 2.2 +/- 0.1 min(-1). The phosphorylated HA is inactive against HA-sensitive Delta tolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k(cat) and K(m) values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5'''-dihydrohygromycin A and 5'''-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.