Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding

SummaryThe LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytR family of transcription factors that regulate virulence factor and toxin gene expression in pathogenic bacteria. This previously uncharacterized domain lacks sequence similarity with proteins of known structure. The crystal structure of the DNA-binding domain of Staphylococcus aureus AgrA complexed with a DNA pentadecamer duplex has been determined at 1.6 Å resolution. The structure establishes a 10-stranded β fold for the LytTR domain and reveals its mode of interaction with DNA. Residues within loop regions of AgrA contact two successive major grooves and the intervening minor groove on one face of the oligonucleotide duplex, inducing a substantial bend in the DNA. Loss of DNA binding upon substitution of key interacting residues in AgrA supports the observed binding mode. This mode of protein-DNA interaction provides a potential target for future antimicrobial drug design

Temperature Regulates Transcription in the Zebrafish Circadian Clock

It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light–dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature

PER-TIM Interactions with the Photoreceptor Cryptochrome Mediate Circadian Temperature Responses in Drosophila

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cryb. The period mutant perL manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of perL flies, because a perL; cryb strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates

Archaeal/Eukaryal RNase P: subunits, functions and RNA diversification

RNase P, a catalytic ribonucleoprotein (RNP), is best known for its role in precursor tRNA processing. Recent discoveries have revealed that eukaryal RNase P is also required for transcription and processing of select non-coding RNAs, thus enmeshing RNase P in an intricate network of machineries required for gene expression. Moreover, the RNase P RNA seems to have been subject to gene duplication, selection and divergence to generate two new catalytic RNPs, RNase MRP and MRP-TERT, which perform novel functions encompassing cell cycle control and stem cell biology. We present new evidence and perspectives on the functional diversification of the RNase P RNA to highlight it as a paradigm for the evolutionary plasticity that underlies the extant broad repertoire of catalytic and unexpected regulatory roles played by RNA-driven RNPs

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Structural characterization of the ribonuclease P protein aRpp29 from the hyperthermophilic sulfate-reducing Archaeon Archaeoglobus fulgidus

The process of tRNA maturation requires extensive posttranscriptional modification. These modifications include 5’ leader removal, 3’ CCA addition, intron splicing, and extensive base modification. The enzyme responsible for the removal of the 5’ leader is known as Ribonuclease P (RNase P). This ribonucleoprotein complex is present in all cells and cellular compartments that perform translation. In this dissertation, the archaeal Ribonuclease P protein aRpp29 from Archaeoglobus fulgidus was structurally characterized using nuclear magnetic resonance (NMR) and X-ray crystallography techniques. The structure of aRpp29 consists of an amino terminal α-helix followed by a six-stranded, anti-parallel β-sheet and then an α-helix at the carboxy terminus. The three dimensional structure forms a semi-closed barrel, wrapped around a well-conserved hydrophobic core. The α-helices align in an anti-parallel orientation, capping the open end of the structure. There are well-conserved charged residues that may present a surface for interactions with either the RNase P RNA or the substrate tRNA. An interesting feature of this structure is an internal salt bridge formed by a triad of conserved residues. This feature may confer the unusual stability observed over a wide range of pH and temperatures. The investigation of the structure of aRpp29 using NMR revealed distinct differences when compared to the structure solved using X-ray crystallography. The solution structure forms the same six stranded anti-parallel β-sheet but lacks stable amino and carboxy terminal helices, indicating that ~40% of the protein is in an equilibrium between a folded and unfolded state. This finding was further investigated by measuring circular dichroism and amide proton exchange rates. The structure of aRpp29 reveals that it is a variant of the Sm-fold (or likeSM) family of proteins. These proteins are involved in a variety of processes involving RNA, including splicing and transcriptional regulation. Sm proteins and their homologs form heptameric rings in solution, although untracentrifugation studies show that aRpp29 forms a monomer in solution. The structural studies of archaeal Ribonuclease P protein Rpp29 presented in this dissertation provide an essential step toward understanding the overall architecture of ribonuclease P.Institute for Cellular and Molecular Biolog

Animal contact and paediatric acute febrile illness in Greater Accra Region, Ghana

To examine the association between animal contact (primarily dogs and cats) and non-malarial fever, as well as with secondary symptoms of headache, nausea, vomiting, and cough, in 687 children in Greater Accra Region, Ghana. Cross-sectional study of acute febrile illness among children aged 1-15 years old between October 2016 and August 2017. Ledzokuku-Krowor Municipal Assembly (LEKMA) Hospital, Teshie, Greater Accra Region. The study included children with acute fever, defined as a measured temperature of greater than 37.5°C, occurring less than seven days before the hospital visit, and afebrile children as controls. Measured fever, self-reported fever, and secondary symptoms, each adjusting for patient household characteristics. Animal contact was neither associated with measured fever (OR = 1.04, 95% CI 0.73-1.49) nor with self-reported fever (OR = 0.97, 95% CI 0.68-1.39). Animal contact was associated with headache (OR = 3.26, 95% CI 2.23-4.77, < .01) and nausea (OR = 3.05, 95% CI 1.99-4.68, < .01), but not with vomiting or cough. Additional models that used alternate inclusion criteria to define non-malarial fever yielded similar results. Several bacterial zoonoses that could plausibly have been transmitted by dogs and cats were diagnosed in the study population. These findings suggest the need for future studies to evaluate animal contact as a risk factor for bacterial zoonoses that may serve as an etiological driver of acute febrile illness. no external funding