Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT.

Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.

Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

Lowering GTP level increases survival of amino acid starvation but slows growth rate for Bacillus subtilis cells lacking (p)ppGpp.

Bacterial cells sense external nutrient availability to regulate macromolecular synthesis and consequently their growth. In the Gram-positive bacterium Bacillus subtilis, the starvation-inducible nucleotide (p)ppGpp negatively regulates GTP levels, both to resist nutritional stress and to maintain GTP homeostasis during growth. Here, we quantitatively investigated the relationship between GTP level, survival of amino acid starvation, and growth rate when GTP synthesis is uncoupled from its major homeostatic regulator, (p)ppGpp. We analyzed growth and nucleotide levels in cells that lack (p)ppGpp and found that their survival of treatment with a nonfunctional amino acid analog negatively correlates with both growth rate and GTP level. Manipulation of GTP levels modulates the exponential growth rate of these cells in a positive dose-dependent manner, such that increasing the GTP level increases growth rate. However, accumulation of GTP levels above a threshold inhibits growth, suggesting a toxic effect. Strikingly, adenine counteracts GTP stress by preventing GTP accumulation in cells lacking (p)ppGpp. Our results emphasize the importance of maintaining appropriate levels of GTP to maximize growth: cells can survive amino acid starvation by decreasing GTP level, which comes at a cost to growth, while (p)ppGpp enables rapid adjustment to nutritional stress by adjusting GTP level, thus maximizing fitness.

GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction.

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction.

Failsafe mechanisms couple division and DNA replication in bacteria.

The past 20 years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly [1]. At the same time, we understand very little about the relationship between cell division and other cell-cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication, followed by a complete arrest in cell growth. Arrested cells are metabolically active but are unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state in which cells are unable to resume growth or division when returned to permissive conditions. Our data suggest the presence of two failsafe mechanisms: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. These findings contradict the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell-cycle arrest validates the bacterial cell-cycle machinery as an effective target for antimicrobial development.