Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy.

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.

Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite.

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum.

Genetic interaction screens with ordered overexpression and deletion clone sets implicate the Escherichia coli GTPase YjeQ in late ribosome biogenesis.

The Escherichia coli protein YjeQ is a circularly permuted GTPase that is broadly conserved in bacteria. An emerging body of evidence, including cofractionation and in vitro binding to the ribosome, altered polysome profiles after YjeQ depletion, and stimulation of GTPase activity by ribosomes, suggests that YjeQ is involved in ribosome function. The growth of strains lacking YjeQ in culture is severely compromised. Here, we probed the cellular function of YjeQ with genetic screens of ordered E. coli genomic libraries for suppressors and enhancers of the slow-growth phenotype of a delta yjeQ strain. Screening for suppressors using an ordered library of 374 clones overexpressing essential genes and genes associated with ribosome function revealed that two GTPases, Era and initiation factor 2, ameliorated the growth and polysome defects of the delta yjeQ strain. In addition, seven bona fide enhancers of slow growth were identified (delta tgt, delta ksgA, delta ssrA, delta rimM, delta rluD, delta trmE/mnmE, and delta trmU/mnmA) among 39 deletions (in genes associated with ribosome function) that we constructed in the delta yjeQ genetic background. Taken in context, our work is most consistent with the hypothesis that YjeQ has a role in late 30S subunit biogenesis.

Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function.

We present an analysis of the cellular phenotype and biochemical activity of a conserved bacterial GTPase of unknown function (YloQ and YjeQ in Bacillus subtilis and Escherichia coli respectively) using a collection of antibiotics of diverse mechanisms and chemical classes. We created a yloQ deletion strain, which exhibited a slow growth phenotype and formed chains of filamentous cells. Additionally, we constructed a conditional mutant in yloQ, where growth was dependent on inducible expression from a complementing copy of the gene. In phenotypic studies, depletion of yloQ sensitized cells to antibiotics that bind at the peptide channel or peptidyl transferase centre, providing the first chemical genetic evidence linking this GTPase to ribosome function. Additional experiments using these small-molecule probes in vitro revealed that aminoglycoside antibiotics severely affected a previously characterized ribosome-associated GTPase activity of purified, recombinant YjeQ from E. coli. None of the antibiotics tested competed with YjeQ for binding to 30 or 70 S ribosomes. A closer examination of YloQ depletion revealed that the polyribosome profiles were altered and that decreased expression of YloQ led to the accumulation of ribosomal subunits at the expense of intact 70 S ribosomes. The present study provides the first evidence showing that YloQ/YjeQ may be involved in several areas of cellular metabolism, including cell division and ribosome function.

Probing the active site of YjeE: a vital Escherichia coli protein of unknown function.

In the study described here, we have taken steps to characterize the YjeE protein, an Escherichia coli protein of unknown function that is essential for bacterial viability. YjeE represents a protein family whose members are broadly conserved in bacteria, absent from eukaryotes and contain both Walker A and B motifs, characteristic of P-loop ATPases. We have revisited the dispensability of the yjeE gene in E. coli and describe efforts to probe the function of the YjeE protein with in vitro biochemistry. We have looked critically for ATPase activity in the recombinant E. coli protein and have made vigilant use of site-directed variants in the Walker A [K41A (Lys41-->Ala) and T42A] and putative Walker B (D80Q) motifs. We noted that any hydrolysis of ATP by the wild-type E. coli protein might be attributed to background ATPase, since it was not appreciably different from that of the variants. To overcome potential contaminants, we turned to crystalline pure YjeE protein from Haemophilus influenzae that was found to hydrolyse ATP at a slow rate (kcat=1 h(-1)). We have also shown high-affinity binding to YjeE by ADP using equilibrium dialysis (K(d)=32 microM) and by fluorescence resonance energy transfer from a conserved tryptophan in YjeE to a fluorescent derivative of ADP, 2'-/3'-O-(N-methylanthraniloyl)adenosine 5'-O-diphosphate (K(d)=8 microM). Walker motif variants were notably impaired for ADP binding and T42A and D80Q mutations in yjeE were incapable of complementing the yjeE deletion strain.

The Apicomplexan AP2 family: integral factors regulating Plasmodium development.

Malaria is caused by protozoan parasites of the genus Plasmodium and involves infection of multiple hosts and cell types during the course of an infection. To complete its complex life cycle the parasite requires strict control of gene regulation for survival and successful propagation. Thus far, the Apicomplexan AP2 (ApiAP2) family of DNA-binding proteins is the sole family of proteins to have surfaced as candidate transcription factors in all apicomplexan species. Work from several laboratories is beginning to shed light on how the ApiAP2 proteins from Plasmodium spp. contribute to the regulation of gene expression at various stages of parasite development. Here we highlight recent progress toward understanding the role of Plasmodium ApiAP2 proteins in DNA related regulatory processes including transcriptional regulation and gene silencing.

The ATPase activity of an 'essential' Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization.

Characterization of 'unknown' proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K(m) of about 60 microM for ATP and a V(max) of about 10 nmol min(-1) (mg protein)(-1) (k(cat) approximately 10 h(-1)). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

Isolation of the rstA gene as a multicopy suppressor of YjeE, an essential ATPase of unknown function in Escherichia coli.

YjeE is an essential ATPase in Escherichia coli whose cellular function remains uncharacterized. Using a genomic library, we have identified rstA as a multicopy suppressor of a conditional yjeE deletion strain. High-copy rstA is the first recorded suppressor for a lesion in yjeE, and this newly charted genetic interaction has the potential to be informative about the function, with further study of the interacting partners.

The yjeQ gene is required for virulence of Staphylococcus aureus.

Gene products required for in vivo growth and survival of Staphylococcus aureus and other pathogens represent new targets for antimicrobial chemotherapy. In this study we created a Staphylococcus aureus yjeQ deletion strain and tested its virulence using a mouse kidney abscess infection model. The yjeQ deletion strain was compromised for growth in vitro and severely attenuated for virulence. We concluded that yjeQ is an attractive and novel new drug target.

Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis.

The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.