Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.

Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S ribosomal subunit to the 40 S ribosomal subunit during translation initiation. Formation of the resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity of the factor for the complex and allowing it to dissociate. Here we present a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathway. Our data indicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translation initiation, including the eIF1-and eIF1A-dependent delivery of initiator methionyl-tRNA to the 40 S ribosomal subunit and subsequent GTP hydrolysis by eIF2. Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which has previously been shown to interact with the C terminus of eIF5B. Disruption of either isoleucine residue in the eIF1A C-terminal sequence DIDDI reduces the rate constant for GTP hydrolysis by ϳ20-fold, whereas changing the aspartic acid residues has no effect. Changing the isoleucines in the C terminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining. These data indicate that the interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly provides a checkpoint for correct complex formation, allowing full activation of GTP hydrolysis only upon formation of a properly organized 80 S initiation complex.The ultimate goal of translation initiation is to assemble a ribosomal complex with an initiator methionyl-tRNA (Met-tRNA i ) 2 positioned at the AUG start codon of the mRNA in the P-site of the ribosome. In bacteria, this process requires three initiation factors (IFs) as well as the hydrolysis of GTP. IF1 directs fMet-tRNA i to the P-site of the small ribosomal subunit by blocking the A-site. IF2 is a GTPase involved in both fMet-tRNA binding and subunit joining. IF3 helps to ensure the fidelity of the mRNA/tRNA interaction in the ribosomal P-site and is ultimately involved in ribosome recycling (1).In eukaryotes, translation initiation is significantly more complex, requiring at least 12 initiation factors (eIFs) and the hydrolysis of both ATP and GTP to assemble an 80 S complex capable of elongation. In the current model (2, 3), translation initiation begins with the formation of an eIF2⅐GTP⅐Met-tRNA i ternary complex (TC). TC is loaded onto the 40 S ribosomal subunit with the help of eIF1, -1A, and -3. The resulting 43 S complex then binds near the 5Ј 7-methylguanosine cap of an mRNA and is thought to scan the mRNA in search of an AUG start codon. Upon AUG recognition, eIF2 hydrolyzes its bound GTP with the help of the GTPase-activating protein eIF5. These events appear to be triggered by an AUG-dependent conformational change in the 43 S⅐mRNA complex that leads to a destabilization of eIF1 binding (4). The movement or dissociation of eIF1 triggers irreversible GTP hydrolysis via the gated release of P i from the complex (5). At this poin...

SummaryLittle is known about the molecular mechanics of the late events of translation initiation in eukaryotes. We present a kinetic dissection of the transition from a pre-initiation complex (PIC) after start codon recognition to the final 80S initiation complex (IC). The resulting framework reveals that eIF5B actually accelerates the rate of ribosomal subunit joining and this acceleration is influenced by the conformation of the GTPase active site of the factor mediated by the bound nucleotide. eIF1A accelerates joining through its C-terminal interaction with eIF5B, and eIF1A release from the initiating ribosome, which occurs only after subunit joining, is accelerated by GTP hydrolysis by eIF5B. Following subunit joining, GTP hydrolysis by eIF5B alters the conformation of the final IC and clears a path to promote rapid release of eIF1A. Our data, coupled with previous work, indicate that eIF1A is present on the ribosome throughout the entire initiation process and plays key roles at every stage.

Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multi-systemic syndromes. Here we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism, and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the γ subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2γ function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms.

In contrast to elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds initiator Met-tRNAiMet to the P-site of the 40S ribosomal subunit. We used directed hydroxyl radical probing experiments to map the binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNAiMet. Our results identify a key binding-interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Moreover, we showed that eIF2γ primarily contacts the acceptor stem of Met-tRNAiMet. Whereas the analogous domain III of EF-Tu contacts the T-stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, but not Met-tRNAiMet, binding. Thus despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.

A purine repressor (PurR) mediates adenine nucleotide-dependent regulation of transcription initiation of the Bacillus subtilis pur operon. This repressor has been purified for the first time, and binding to control site DNA was characterized. PurR binds in vitro to four operons. Apparent K d values for binding were 7 nM for the pur operon, 8 nM for purA, 13 nM for purR, and 44 nM for the pyr operon. In each case, DNase I footprints exhibited a pattern of protected and hypersensitive sites that extended over more than 60 bp. A GAAC-N 24 -GTTC sequence in the pur operon was necessary but not sufficient for the PurR-DNA interaction. However, this motif, which is conserved in the four binding sites, was not required for binding of PurR to purA. Thus, the common DNA recognition element for binding of PurR to the four operons is not known. Multiple PurR-pur operon DNA complexes having a binding stoichiometry that was either approximately two or six repressor molecules per DNA fragment were detected. The results of a torsional constraint experiment suggest that control site DNA forms one right-handed turn around PurR.The genes required for de novo synthesis of IMP are clustered in a 12-gene polycistronic operon in Bacillus subtilis (10). Transcription of the pur operon is subject to dual regulation. The addition of adenine to cells results in the repression of transcription initiation, whereas the addition of guanine signals premature transcription termination in the 242-nucleotide (nt) mRNA leader region preceding the first gene of the operon. A purine repressor (PurR) is required for the regulation of transcription initiation (11). The repressor is a 62-kDa homodimer containing 285 amino acid subunits (31) encoded by a purR gene at about 6°in a sequenced region (19) of the B. subtilis chromosome. The pur operon DNA site to which the crude repressor bound was mapped to a position corresponding to approximately Ϫ136 to Ϫ26 relative to the start of transcription (11). This site is contiguous with and overlaps the Ϫ35 promoter element at nts Ϫ33 to Ϫ28. Binding of PurR to the pur operon control site was blocked by phosphoribosylpyrophosphate (PRPP) leading to a model in which the excess adenine signal is transmitted to PurR by the PRPP pool (31). It was proposed that upon uptake, adenine is converted to adenine 5Ј nucleotides and that the resulting allosteric inhibition of PRPP synthetase by ADP (1) lowers the PRPP pool levels (26), permitting PurR to bind to and repress the transcription of the pur operon. B. subtilis PurR bears no amino acid sequence similarity to Escherichia coli PurR. Furthermore, there is no similarity in the DNA control sites for these two repressors or in the purine or purine nucleotide signals that modulate binding to the DNA control sites.There is presently no information about what determines B. subtilis PurR-pur operon DNA binding specificity. It has been noted, however, that PurR also binds to purR and to purA (31), but the common recognition determinant is unknown. The purA gene encodes the...