Structural analysis of noncanonical translation initiation complexes.

Translation initiation is a highly regulated, multi-step process which is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of E. coli 70S initiation complexes containing an fMet-tRNAfMet G30-C40 variant paired to noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact start codon selection.

Ratchet, swivel, tilt and roll: a complete description of subunit rotation in the ribosome.

Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.

Recent advances in infectious disease research using cryo-electron tomography.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

How Heme Oxygenase-1 Prevents Heme-Induced Cell Death.

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by "loose" (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity--CO and bilirubin--have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity.