We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

https://science.sciencemag.org/content/sci/292/5518/883.full.pdf

Crystal structure of the ribosome at 5.5 A resolution

Antibiotic drug-target interactions, and their respective direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochemical pathways. In this Review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, could be exploited to create new antibacterial therapies.

How antibiotics kill bacteria: from targets to networks

MAP kinases are among the most ancient signal transduction pathways and are widely used throughout evolution in many physiological processes. In mammalian species, MAP kinases are involved in all aspects of immune responses, from the initiation phase of innate immunity, to activation of adaptive immunity, and to cell death when immune function is complete. In this review, we summarize recent progress in understanding the function and regulation of MAP kinase pathways in these phases of immune responses.

MAP Kinases in the Immune Response

Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.

/pdf/adaptive-recognition-by-nucleic-acid-aptamers-3atike4jl7.pdf

Adaptive recognition by nucleic acid aptamers.

After encountering antigen, helper T (T(H)) cells undergo differentiation to effector cells, which can secrete high levels of interferon-gamma, interleukin-4 (IL-4), IL-10 and other immunomodulators. How T(H) cells acquire, and remember, new patterns of gene expression is an area of intensive investigation. The process is remarkably plastic, with cytokines being key regulators. Extrinsic signals seem to be integrated into cell-intrinsic programming, in what is becoming an intriguing story of regulated development. We summarize the latest insights into mechanisms that govern the lineage choices that are made during T(H)-cell responses to foreign pathogens.

/pdf/the-lineage-decisions-of-helper-t-cells-5g789x1rf0.pdf

The lineage decisions of helper T cells

Aminoglycoside antibiotics that bind to 30S ribosomal A-site RNA cause misreading of the genetic code and inhibit translocation. The aminoglycoside antibiotic paromomycin binds specifically to an RNA oligonucleotide that contains the 30S subunit A site, and the solution structure of the RNA-paromomycin complex was determined by nuclear magnetic resonance spectroscopy. The antibiotic binds in the major groove of the model A-site RNA within a pocket created by an A-A base pair and a single bulged adenine. Specific interactions occur between aminoglycoside chemical groups important for antibiotic activity and conserved nucleotides in the RNA. The structure explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms, and various resistance mechanisms, and provides insight into ribosome function.

https://science.sciencemag.org/content/sci/274/5291/1367.full.pdf

Structure of the A Site of Escherichia coli 16S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic

Signal transduction via MAP kinase pathways plays a key role in a variety of cellular responses, including growth factor-induced proliferation, differentiation and cell death. In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro-inflammatory cytokines and environmental stress. Although p38 MAP kinase is implicated in the control of inflammatory responses, the molecular mechanisms remain unclear. Upon activation, CD4+ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro-inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds (specific inhibitors of p38 MAP kinase) block the production of interferon-gamma (IFNgamma) by Th1 cells without affecting IL-4 production by Th2 cells. These drugs also inhibit transcription driven by the IFNgamma promoter. In transgenic mice, inhibition of the p38 MAP kinase pathway by the expression of dominant-negative p38 MAP kinase results in selective impairment of Th1 responses. In contrast, activation of the p38 MAP kinase pathway by the expression of constitutivelyactivated MAP kinase kinase 6 in transgenic mice caused increased production of IFNgamma during the differentiation and activation of Th1 cells. Together, these data demonstrate that the p38 MAP kinase is relevant for Th1 cells, not Th2 cells, and that inhibition of p38 MAP kinase represents a possible site of therapeutic intervention in diseases where a predominant Th1 immune response leads to a pathological outcome. Moreover, our study provides an additional mechanism by which the p38 MAP kinase pathway controls inflammatory responses.

/pdf/interferon-g-expression-by-th1-effector-t-cells-mediated-by-3z98s59dol.pdf

Interferon‐γ expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway

RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 s rRNA

The aminoglycosides, a group of structurally related antibiotics, bind to rRNA in the small subunit of the prokaryotic ribosome. Most aminoglycosides are inactive or weakly active against eukaryotic ribosomes. A major difference in the binding site for these antibiotics between prokaryotic and eukaryotic ribosomes is the identity of the nucleotide at position 1408 ( Escherichia coli numbering), which is an adenosine in prokaryotic ribosomes and a guanosine in eukaryotic ribosomes. Expression in E.coli of plasmid‐encoded 16S rRNA containing an A1408 to G substitution confers resistance to a subclass of the aminoglycoside antibiotics that contain a 6′ amino group on ring I. Chemical footprinting experiments indicate that resistance arises from the lower affinity of the drug for the eukaryotic rRNA sequence. The 1408G ribosomes are resistant to the same subclass of aminoglycosides as previously observed both for eukaryotic ribosomes and bacterial ribosomes containing a methylation at the N1 position of A1408. The results indicate that the identity of the nucleotide at position 1408 is a major determinant of specificity of aminoglycoside action, and agree with prior structural studies of aminoglycoside–rRNA complexes.

/pdf/basis-for-prokaryotic-specificity-of-action-of-2y0j419e4c.pdf

Michael I. Recht

Papers

Structure of the A Site of Escherichia coli 16S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic

Interferon‐γ expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway

RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

Binding of neomycin-class aminoglycoside antibiotics to the A-site of 16 s rRNA

Basis for prokaryotic specificity of action of aminoglycoside antibiotics.