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 39 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.

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 39 end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.

Bacterial protein synthesis is an essential, conserved, and environmentally responsive process. Yet, many of its components and dependencies remain unidentified. To address this gap, we used quantitative synthetic genetic arrays to map functional relationships among >48,000 gene pairs in Escherichia coli under four culture conditions differing in temperature and nutrient availability. The resulting data provide global functional insights into the roles and associations of genes, pathways, and processes important for efficient translation, growth, and environmental adaptation. We predict and independently verify the requirement of unannotated genes for normal translation, including a previously unappreciated role of YhbY in 30S biogenesis. Dynamic changes in the patterns of genetic dependencies across the four growth conditions and data projections onto other species reveal overarching functional and evolutionary pressures impacting the translation system and bacterial fitness, underscoring the utility of systematic screens for investigating protein synthesis, adaptation, and evolution.

Mainstream information security awareness techniques are failing to evolve at the same rate as automated technical security controls. Humans are increasingly seen as the weak link in information security defences and attackers are starting to prefer exploiting human factors such as greed, curiosity and respect for authority. Problems with human behaviour in an information security context are assumed to be caused by a lack of facts available to the audience. Awareness therefore is largely treated as the broadcast of facts to an audience in the hope that behaviour improves. There is a tendency for technical experts in the field of information security to tell people what they think they ought to know (and may in fact already know). This "technocratic" view of risk communication is fundamentally flawed and has been strongly criticised by experts in safety risk communications as ineffective and inefficient. To improve the effectiveness and efficiency of security awareness techniques this paper leverages safety risk communications which is a mature discipline with common objectives. A critical feature of safety risk communications which is missing from the information security approach is a set of methodologies to systematically evaluate audience requirements. Accordingly, this paper explores the concepts of bounded rationality, mental models and the Extended Parallel Processing Model in an information security context.