RNase III is a global regulator of gene expression in Escherichia coli that is instrumental in the maturation of ribosomal and other structural RNAs. We examine here how RNase III itself is regulated in response to growth and other environmental changes encountered by the cell and how, by binding or processing double-stranded RNA (dsRNA) intermediates, RNase III controls the expression of genes. Recent insight into the mechanism of dsRNA binding and processing, gained from structural studies of RNase III, is reviewed. Structural studies also reveal new cleavage sites in the enzyme that can generate longer 3′ overhangs.

In addition to storage of genetic information, DNA can also catalyze various reactions. RNA-cleaving DNAzymes are the catalytic DNAs discovered the earliest, and they can cleave RNAs in a sequence-specific manner. Owing to their great potential in medical therapeutics, virus control, and gene silencing for disease treatments, RNA-cleaving DNAzymes have been extensively studied; however, the mechanistic understandings of their substrate recognition and catalysis remain elusive. Here, we report three catalytic form 8–17 DNAzyme crystal structures. 8–17 DNAzyme adopts a V-shape fold, and the Pb²⁺ cofactor is bound at the pre-organized pocket. The structures with Pb²⁺ and the modification at the cleavage site captured the pre-catalytic state of the RNA cleavage reaction, illustrating the unexpected Pb²⁺-accelerated catalysis, intrinsic tertiary interactions, and molecular kink at the active site. Our studies reveal that DNA is capable of forming a compacted structure and that the functionality-limited bio-polymer can have a novel solution for a functional need in catalysis.

As the largest group of MYB family transcription factors, R2R3-MYB proteins play essential roles during plant growth and development. However, the structural basis underlying how R2R3-MYBs recognize the target DNA remains elusive. Here, we report the crystal structure of Arabidopsis WEREWOLF (WER), an R2R3-MYB protein, in complex with its target DNA. Structural analysis showed that the third α-helices in both the R2 and R3 repeats of WER fit in the major groove of the DNA, specifically recognizing the DNA motif 5′-AACNGC-3′. In combination with mutagenesis, in vitro binding and in vivo luciferase assays, we showed that K55, N106, K109 and N110 are critical for the function of WER. Although L59 of WER is not involved in DNA binding in the structure, ITC analysis suggested that L59 plays an important role in sensing DNA methylation at the fifth position of cytosine (5mC). Like 5mC, methylation at the sixth position of adenine (6mA) in the AAC element also inhibits the interaction between WER and its target DNA. Our study not only unravels the molecular basis of how WER recognizes its target DNA, but also suggests that 5mC and 6mA modifications may block the interaction between R2R3-MYB transcription factors and their target genes.

Although quorum-sensing (QS) systems are important regulators of virulence gene expression in the opportunistic human pathogen Pseudomonas aeruginosa, their detailed regulatory mechanisms have not been fully characterized. Here, we show that deletion of PA2588 resulted in increased production of pyocyanin and biofilm, as well as enhanced pathogenicity in a mouse model. To gain insights into the function of PA2588, we performed a ChIP-seq assay and identified 28 targets of PA2588, including the intergenic region between PA2588 and pqsH, which encodes the key synthase of Pseudomonas quinolone signal (PQS). Though the C-terminal domain was similar to DNA-binding regions of other AraC family members, structural studies revealed that PA2588 has a novel fold at the N-terminal region (NTR), and its C-terminal HTH (helix-turn-helix) domain is also unique in DNA recognition. We also demonstrated that the adaptor protein ClpS, an essential regulator of ATP-dependent protease ClpAP, directly interacted with PA2588 before delivering CdpR to ClpAP for degradation. We named PA2588 as CdpR (ClpAP-degradation and pathogenicity Regulator). Moreover, deletion of clpP or clpS/clpA promotes bacterial survival in a mouse model of acute pneumonia infection. Taken together, this study uncovered that CdpR is an important QS regulator, which can interact with the ClpAS-P system to regulate the expression of virulence factors and pathogenicity. Although many transcriptional regulators tune P. aeruginosa virulence factor expression and secretion, the molecular mechanisms of the underlying regulatory network are still elusive. Quorum sensing, the ability of bacteria to communicate and detect cell density to determine the most advantageous time to orchestrate collective events, is known to govern P. aeruginosa virulence. In this study, we present a novel AraC-family transcription factor, CdpR (PA2588), that controls numerous virulence factors via directly regulating the quorum sensing Pseudomonas quinolone signal (PQS) system. We solved the crystal structure of CdpR, which showed that its N-terminal domain contains a unique fold that is different from other AraC-family proteins. In addition, we found that CdpR is regulated by the ClpAS-ClpP protease. CdpR interacts with the adaptor protein ClpS and then is degraded by the ATP-dependent ClpAP protease. This is the first example of a quorum-sensing regulator as a substrate of the ClpAS-ClpP protease in P. aeruginosa. These findings significantly extend our understandings of quorum sensing and virulence regulation and provide insights into the function of proteases in pathogenic bacteria.