Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways.

In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.

Structure and dynamics study of translation initiation factor 1 from Staphylococcus aureus suggests its RNA binding mode.

Translation initiation, the rate-limiting step in the protein synthesis, is tightly regulated. As one of the translation initiation factors, translation initiation factor 1 (IF1) plays crucial roles not only in translation but also in many cellular processes that are important for genomic stability, such as the activity of RNA chaperones. Here, we characterize the RNA interactions and dynamics of IF1 from Staphylococcus aureus Mu50 (IF1Sa) by NMR spectroscopy. First, the NMR-derived solution structure of IF1Sa revealed that IF1Sa adopts an oligonucleotide/oligosaccharide binding (OB)-fold. Structural comparisons showed large deviations in the α-helix and the following loop, which are potential RNA-binding regions of the OB-fold, as well as differences in the electrostatic potential surface among bacterial IF1s. Second, the 15N NMR relaxation data for IF1Sa indicated the flexible nature of the α-helix and the following loop region of IF1Sa. Third, RNA-binding properties were studied using FP assays and NMR titrations. FP binding assays revealed that IF1Sa binds to RNAs with moderate affinity. In combination with the structural analysis, the NMR titration results revealed the RNA binding sites. Taken together, these results show that IF1Sa binds RNAs with moderate binding affinity via the residues that occupy the large surface area of its ß-barrel. These findings suggest that IF1Sa is likely to bind RNA in various conformations rather than only at a specific site and indicate that the flexible RNA binding mode of IF1Sa is necessary for its interaction with various RNA substrates.

(1)H, (13)C and (15)N resonance assignments and secondary structure analysis of translation initiation factor 1 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of infection in humans. P. aeruginosa can acquire resistance against multiple groups of antimicrobial agents, including ß-lactams, aminoglycosides and fluoroquinolones, and multidrug resistance is increasing in this organism which makes treatment of the infections difficult and expensive. This has led to the unmet need for discovery of new compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Translation initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis, and its structure is unknown. Here we report the (1)H, (13)C and (15)N chemical shift assignments of Pa-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified five ß-strands with an unusually extended ß-strand at the C-terminal end of the protein and one short α-helix arranged in the sequential order ß1-ß2-ß3-α1-ß4-ß5. This is further supported by (15)N-{(1)H} hetero NOEs. These secondary structure elements suggest the Pa-IF1 adopts the typical ß-barrel structure and is composed of an oligomer-binding motif.

Adaptation of Extremophilic Proteins with Temperature and Pressure: Evidence from Initiation Factor 6.

In this work, we study dynamical properties of an extremophilic protein, Initiation Factor 6 (IF6), produced by the archeabacterium Methanocaldococcus jannascii, which thrives close to deep-sea hydrothermal vents where temperatures reach 80 °C and the pressure is up to 750 bar. Molecular dynamics simulations (MD) and quasi-elastic neutron scattering (QENS) measurements give new insights into the dynamical properties of this protein with respect to its eukaryotic and mesophilic homologue. Results obtained by MD are supported by QENS data and are interpreted within the framework of a fractional Brownian dynamics model for the characterization of protein relaxation dynamics. IF6 from M. jannaschii at high temperature and pressure shares similar flexibility with its eukaryotic homologue from S. cerevisieae under ambient conditions. This work shows for the first time, to our knowledge, that the very common pattern of corresponding states for thermophilic protein adaptation can be extended to thermo-barophilic proteins. A detailed analysis of dynamic properties and of local structural fluctuations reveals a complex pattern for "corresponding" structural flexibilities. In particular, in the case of IF6, the latter seems to be strongly related to the entropic contribution given by an additional, C-terminal, 20 amino-acid tail which is evolutionary conserved in all mesophilic IF6s.

The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli.

LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine-Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.

Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro.

Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant T7 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg(2+)-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection.

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution.

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.

Crystal structure of TTHA0061, an uncharacterized protein from Thermus thermophilus HB8, reveals a novel fold.

The crystal structure of an uncharacterized protein TTHA0061 from Thermus thermophilus HB8, was determined and refined to 1.8 A by a single wavelength anomalous dispersion (SAD) method. The structural analysis and comparison of TTHA0061 with other existing structures in the Protein Data Bank (PDB) revealed a novel fold, suggesting that this protein may belong to a translation initiation factor or ribosomal protein family. Differential scanning calorimetry analysis suggested that the thermostability of TTHA0061 increased at pH ranges of 5.8-6.2, perhaps due to the abundance of glutamic acid residues.

Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya.

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal-eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.

Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria.

Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), initiator fMet-tRNA(fMet), three initiation factors, IF1, IF2, and IF3, and the large ribosomal subunit (50S). During initiation, the 30S subunit, in a complex with IF3, binds mRNA, IF1, IF2.GTP, and fMet-tRNA(fMet) to form a 30S initiation complex which then recruits the 50S subunit to yield a 70S initiation complex, while the initiation factors are released. Here we describe a transient kinetic approach to study the timing of elemental steps of 30S initiation complex formation, 50S subunit joining, and the dissociation of the initiation factors from the 70S initiation complex. Labeling of ribosomal subunits, fMet-tRNA(fMet), mRNA, and initiation factors with fluorescent reporter groups allows for the direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer (FRET) between two fluorophores. Subunit joining was monitored by light scattering or by FRET between dyes attached to the ribosomal subunits. The kinetics of chemical steps, that is, GTP hydrolysis by IF2 and peptide bond formation following the binding of aminoacyl-tRNA to the 70S initiation complex, were measured by the quench-flow technique. The methods described here are based on results obtained with initiation components from Escherichia coli but can be adopted for mechanistic studies of initiation in other prokaryotic or eukaryotic systems.

The GTPase, CpgA(YloQ), a putative translation factor, is implicated in morphogenesis in Bacillus subtilis.

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or 'curly' shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance.

Selenocysteine tRNA-specific elongation factor SelB is a structural chimaera of elongation and initiation factors.

In all three kingdoms of life, SelB is a specialized translation elongation factor responsible for the cotranslational incorporation of selenocysteine into proteins by recoding of a UGA stop codon in the presence of a downstream mRNA hairpin loop. Here, we present the X-ray structures of SelB from the archaeon Methanococcus maripaludis in the apo-, GDP- and GppNHp-bound form and use mutational analysis to investigate the role of individual amino acids in its aminoacyl-binding pocket. All three SelB structures reveal an EF-Tu:GTP-like domain arrangement. Upon binding of the GTP analogue GppNHp, a conformational change of the Switch 2 region in the GTPase domain leads to the exposure of SelB residues involved in clamping the 5' phosphate of the tRNA. A conserved extended loop in domain III of SelB may be responsible for specific interactions with tRNA(Sec) and act as a ruler for measuring the extra long acceptor arm. Domain IV of SelB adopts a beta barrel fold and is flexibly tethered to domain III. The overall domain arrangement of SelB resembles a 'chalice' observed so far only for initiation factor IF2/eIF5B. In our model of SelB bound to the ribosome, domain IV points towards the 3' mRNA entrance cleft ready to interact with the downstream secondary structure element.

Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV.

Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 degrees C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-A resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.