In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.

The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase‐like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase‐Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.

Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains unresolved. We show that Stag2 deletion in hematopoietic stem and progenitor cells (HSPCs) results in altered hematopoietic function, increased selfrenewal, and impaired differentiation. Chromatin immunoprecipitation (ChIP) sequencing revealed that, although Stag2 and Stag1 bind a shared set of genomic loci, a component of Stag2 binding sites is unoccupied by Stag1, even in Stag2-deficient HSPCs. Although concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to increased self-renewal and reduced HSPC commitment to the B cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.

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SummaryThe SmpB-tmRNA-mediated trans-translation system has two well-established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non-stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active transtranslation in facilitating RNase R engagement and promoting non-stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non-stop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA-facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans-translation-mediated non-stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.

SsrA is a versatile RNA molecule found in all bacteria that functions as both a tRNA and an mRNA. SsrA rescues ribosomes stalled on damaged mRNAs and directs the tagging and degradation of their aberrant protein products. Small protein B (SmpB) is required for all known activities of SsrA. The two known functions of SmpB are binding SsrA RNA and promoting stable association of the SmpB⅐SsrA complex with 70S ribosomes. Using mutational analysis and biochemical experiments, we have discovered a previously uncharacterized SmpB function. This function is required for a step in the tagging process downstream of SsrA binding and ribosome association but before transpeptidation of the SsrA-linked alanine and establishment of the SsrA reading frame. Our results clearly demonstrate that residues in the C-terminal tail of SmpB confer a hitherto unrevealed function that is essential for trans-translation. Based on these results, we propose that upon binding stalled ribosomes, the unstructured C-terminal tail of SmpB acquires contacts that are critical for productive accommodation of SsrA into the ribosomal A site.SsrA ͉ translation

Yersinia utilizes a sophisticated type III secretion system to enhance its chances of survival and to overcome the host immune system. SmpB (small protein B) and SsrA (small stable RNA A) are components of a unique bacterial translational control system that help maintain the bacterial translational machinery in a fully operational state. We have found that loss of the SmpB-SsrA function causes acute defects in the ability of Yersinia pseudotuberculosis to survive in hostile environments. Most significantly, we show that mutations in smpB-ssrA genes render the bacterium avirulent and unable to cause mortality in mice. Consistent with these observations, we show that the mutant strain is unable to proliferate in macrophages and exhibits delayed Yop-mediated host cell cytotoxicity. Correspondingly, we demonstrate that the smpB-ssrA mutant suffers severe deficiencies in expression and secretion of Yersinia virulence effector proteins, and that this defect is at the level of transcription. Of further interest is the finding that the SmpB-SsrA system might play a similar role in the related type III secretion system that governs flagella assembly and bacterial motility. These findings highlight the significance of the SmpB-SsrA system in bacterial pathogenesis, survival under adverse environmental conditions, and motility.