Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii

Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability.

Structures of tmRNA and SmpB as they transit through the ribosome

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.

Ribosome Rescue Pathways in Bacteria

Ribosomes that become stalled on truncated or damaged mRNAs during protein synthesis must be rescued for the cell to survive. Bacteria have evolved a diverse array of rescue pathways to remove the stalled ribosomes from the aberrant mRNA and return them to the free pool of actively translating ribosomes. In addition, some of these pathways target the damaged mRNA and the incomplete nascent polypeptide chain for degradation. This review highlights the recent developments in our mechanistic understanding of bacterial ribosomal rescue systems, including drop-off, trans-translation mediated by transfer-messenger RNA and small protein B, ribosome rescue by the alternative rescue factors ArfA and ArfB, as well as Bacillus ribosome rescue factor A, an additional rescue system found in some Gram-positive bacteria, such as Bacillus subtilis. Finally, we discuss the recent findings of ribosome-associated quality control in particular bacterial lineages mediated by RqcH and RqcP. The importance of rescue pathways for bacterial survival suggests they may represent novel targets for the development of new antimicrobial agents against multi-drug resistant pathogenic bacteria.

How a circularized tmRNA moves through the ribosome

During trans-translation, transfer-messenger RNA (tmRNA) and small protein B (SmpB) together rescue ribosomes stalled on a truncated mRNA and tag the nascent polypeptide for degradation. We used cryo–electron microscopy to determine the structures of three key states of the tmRNA-SmpB-ribosome complex during trans translation at resolutions of 3.7 to 4.4 angstroms. The results show how tmRNA and SmpB act specifically on stalled ribosomes and how the circularized complex moves through the ribosome, enabling translation to switch from the old defective message to the reading frame on tmRNA.