Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation.

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm , should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X-ray crystallography and cryo-electron microscopy, screening for small-molecule binding partners and comparison of effects of point mutations.

The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli.

When Escherichia coli encounters stress, the endoribonuclease MazF initiates a post-transcriptional response that results in the reprogramming of protein synthesis. By removing the 3΄-terminus of the 16S rRNA, MazF generates specialized ribosomes that selectively translate mRNAs likewise processed by MazF. Given the energy required for de novo ribosome biosynthesis, we considered the existence of a repair mechanism operating upon stress relief to recycle the modified ribosomes. Here, we show that the stress-ribosomes and the 3΄-terminal 16S rRNA fragment are stable during adverse conditions. Moreover, employing in vitro and in vivo approaches we demonstrate that the RNA ligase RtcB catalyzes the re-ligation of the truncated 16S rRNA present in specialized ribosomes Thereby their ability to translate canonical mRNAs is fully restored. Together, our findings not only provide a physiological function for the RNA ligase RtcB in bacteria but highlight the reversibility of ribosome heterogeneity, a crucial but hitherto undescribed concept for translational regulation.

Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli.

The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12.

The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli.

Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation.

Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation.

UNLABELLED: Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) extracellular death factor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS. IMPORTANCE: Bacteria communicate with one another via quorum-sensing (QS) signal molecules. QS provides a mechanism for bacteria to monitor each other's presence and to modulate gene expression in response to population density. Previously, we added E. coli pentapeptide EDF to this list of QS molecules. We showed that, under stress conditions, the induced MazF, an endoribonuclease cleaving at ACA sites, generates EDF from zwf. Here we studied the mechanism of EDF generation and asked whether it is related to EDF density dependency. We illustrated that, under stress conditions, multiple distinct complex mechanisms are involved in EDF generation. This includes formation of leaderless truncated zwf mRNA by MazF, configuration of a length corresponding to the nascent ribosome peptide exit tunnel, rescue performed by the trans-translation system, and cleavage by ClpPX protease. trans-Translation is described as a regulatory system attenuating EDF generation and leading to low levels of EDF in the single cell, as expected for QS.

Ribosome heterogeneity: another level of complexity in bacterial translation regulation.

Translation of the mRNA-encoded genetic information into proteins is catalyzed by the intricate ribonucleoprotein machine, the ribosome. Historically, the bacterial ribosome is viewed as an unchangeable entity, constantly equipped with the entire complement of RNAs and proteins. Conversely, several lines of evidence indicate the presence of functional selective ribosomal subpopulations that exhibit variations in the RNA or the protein components and modulate the translational program in response to environmental changes. Here, we summarize these findings, which raise the functional status of the ribosome from a protein synthesis machinery only to a regulatory hub that integrates environmental cues in the process of protein synthesis, thereby adding an additional level of complexity to the regulation of gene expression.

Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli.

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.

New features of the ribosome and ribosomal inhibitors: non-enzymatic recycling, misreading and back-translocation.

We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome.

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).

[Ribosome recycling revisited].

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G and initiation factor IF3 to disassemble the post-termination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at high resolution reveals the nature and details of the interactions between this protein factor and rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nuleotides of the 23S rRNA, explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling.