RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA

Proteins S4, S16/S17 and S20 of the 30 S ribosomal subunit of Escherichia coli+ associate with specific binding sites in the 16 S ribosomal RNA. A systematic investigation of the co-operative interactions that occur when two or more of these proteins simultaneously attach to the 16 S RNA indicate that their binding sites lie near to one another. The binding site for S4 has previously been located within a 550-nucleotide RNA fragment of approximately 9 S that arises from the 5′-terminal portion of the 16 S RNA upon limited hydrolysis with pancreatic ribonuclease. The 9 S RNA was unable to associate with S20 and S16/S17, however, either alone or in combination. A fragment of similar size and nucleotide sequence, termed the 9 S¹ RNA, has been isolated following ribonuclease digestion of the complex of 16 S RNA with S20 and S16/S17. The 9 S¹ RNA bound not only S4, but S20 and S16/S17 as well, although the fragment complex was stable only when both of the latter protein fractions were present together. Nonetheless, measurements of binding stoichiometry demonstrated the interactions to be specific under these conditions. A comparison of the 9 S and 9 S¹ RNAs by electrophoresis in polyacrylamide gels containing urea revealed that the two fragments differ substantially in the number and distribution of hidden breaks. Contrary to expectation, the RNA in the ribonucleoprotein complex appeared to be more accessible to ribonuclease than the free 16 S RNA as judged by the smaller average length of the sub-fragments recovered from the 9 S¹ RNA. These results suggest that the binding of S4, S16/S17 and S20 brings about a conformational alteration within the 5′ third of the 16 S RNA.To delineate further the portions of the RNA chain that interact with S4, S16/S17 and S20, specific fragments encompassing subsequences from the 5′ third of the 16 S RNA were sought. Two such fragments, designated 12 S-I and 12 S-II, were purified by polyacrylamide gel electrophoresis from partial T₁ ribonuclease digests of the 16 S RNA. The two RNAs, which contain 290 and 210 nucleotides, respectively, are contiguous and together span the entire 5′-terminal 500 residues of the 16 S RNA molecule. When tested individually, neither 12 S-I nor 12 S-II bound S4, S16/S17 or S20. If heated together at 40 °C in the presence of Mg²⁺ ions, however, the two fragments together formed an 8 S complex which associated with S4 alone, with S16/S17 + S20 in combination, and with S4 + S16/S17 + S20 when incubated with an un fractionated mixture of 30 S subunit proteins. These results imply that each fragment contains part of the corresponding binding sites.     

Antimicrobial Use and Resistance in Swine Waste Treatment Systems

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLS_B), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLS_B resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLS_B resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further.     

The endocannabinoid system controls key epileptogenic circuits in the hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.     

Substrate Binding and Active Site Residues in RNases E and G: ROLE OF THE 5��-SENSOR*

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5′-sensor, the S1 domain, and the Mg⁺²/Mn⁺² binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k_cat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.     

Molecular Ecology Of Macrolide–Lincosamide–Streptogramin B Methylases in Waste Lagoons and Subsurface Waters Associated with Swine Production

RNA methylase genes are common antibiotic resistance determinants for multiple drugs of the macrolide, lincosamide, and streptogramin B (MLS_B) families. We used molecular methods to investigate the diversity, distribution, and abundance of MLS_B methylases in waste lagoons and groundwater wells at two swine farms with a history of tylosin (a macrolide antibiotic structurally related to erythromycin) and tetracycline usage. Phylogenetic analysis guided primer design for quantification of MLS_B resistance genes found in tylosin-producing Streptomyces (tlr(B), tlr(D)) and commensal/pathogenic bacteria (erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q)). The near absence of tlr genes at these sites suggested a lack of native antibiotic-producing organisms. The gene combination erm(ABCF) was found in all lagoon samples analyzed. These four genes were also detected with high frequency in wells previously found to be contaminated by lagoon leakage. A weak correlation was found between the distribution of erm genes and previously reported patterns of tetracycline resistance determinants, suggesting that dissemination of these genes into the environment is not necessarily linked. Considerations of gene origins in history (i.e., phylogeny) and gene distributions in the landscape provide a useful “molecular ecology” framework for studying environmental spread of antibiotic resistance.     

Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.The cartilage of the mammalian skeletal system has two distinct roles. The epiphyseal cartilage of the growth plate drives endochondral bone growth, and the hyaline cartilage at the weight-bearing surfaces of bones facilitates joint articulation. In both environments, chondrocyte-regulated production, assembly, and turnover of the extracellular matrix (ECM)¹ are essential for the tissue to withstand compressive forces and respond to mechanical loading. The major structural constituents of cartilage ECM are the heterotypic collagen II/IX/XI fibrils and proteoglycan-glycosaminoglycan networks of aggrecan and hyaluronan. Loss of joint function in osteoarthritis (OA) is strongly associated with net loss of aggrecan and collagen breakdown caused by an imbalance of ECM homeostasis (1). In addition, many inherited human chondrodysplasias involve disruption of cartilage matrix assembly or cell-matrix interactions, resulting in abnormal skeletal development and in some cases early onset cartilage degeneration (2, 3).The alterations in chondrocyte metabolism that occur during OA are complex and remain poorly understood (4). An early response to loss or fragmentation of ECM components is attempted tissue repair through secretion of anabolic factors, cell proliferation, and matrix remodeling (5). However, the resulting product is a fibrocartilage that does not recapitulate the composition or precise architecture of the original hyaline articular cartilage. This limited capacity of cartilage for regeneration has driven research into cartilage tissue engineering (6). Production of authentic hyaline cartilage in vitro remains challenging due to the dedifferentiation of primary chondrocytes upon removal from their three-dimensional matrix environment (7). However, improved “neocartilage” culture systems have been developed through evaluation of suitable chondroprogenitor or chondrocyte subpopulations and optimization of exogenous support matrices and growth factors (8, 9). The therapeutic target of neocartilage culture is autologous tissue repair. However, there is fundamental value in using neocartilage systems to elucidate mechanisms of protein integration into the ECM and the role of specific protein interactions during cartilage maturation.Cartilage profiling by 2-DE and mass spectrometry-based proteomics is generating important new insight into mechanisms of cartilage degeneration in vitro and in vivo (10). For example, anabolic factors with potential roles in cartilage repair, including connective tissue growth factor and inhibin βA (activin), were identified in the secretome of human OA cartilage explants (11). Comparison of cartilage protein extracts from normal donors and OA patients revealed significantly increased levels of the serine protease Htra1 in patient cartilage (12) and that Htra1-mediated proteolysis of aggrecan may significantly contribute to OA pathology (13). Targeted analysis of the chondrocyte mitochondrial proteome highlighted OA-related changes in energy production and protection against reactive oxygen species (14). Obtaining sufficient chondrocytes from human donors for proteomics unfortunately requires expansion of the cell population with potential loss of the chondrocyte phenotype during prolonged culture. Other drawbacks encountered with human samples include the clinical heterogeneity of OA, lack of matched controls, and inherent genetic variation of human subjects (15). Alternatively, animal models that recapitulate hallmarks of progressive cartilage degeneration, such as aggrecan loss and articular surface fibrillation, are emerging as a powerful resource, particularly in mice lacking specific proteases or protease target sites (16, 17). The development of techniques for analysis of murine cartilage using proteomics has paved the way for differential analysis of normal and pathological or genetically targeted cartilage (18, 19).Label-free methods for relative peptide quantitation, such as ion intensity measurement and spectral counting, are emerging as reliable and cost-effective alternatives to chemical modification or isotopic peptide labeling (20). Combining orthogonal protein and/or peptide fractionation with high resolution HPLC-MS can achieve proteome-wide coverage (21). Because extensive sample fractionation can introduce redundancy and variation, improved sequence/proteome coverage must be balanced against the cost of additional sample handling and lengthy LC-MS runs (22).Here we describe a novel platform for analysis of mouse cartilage using solubility-based protein fractionation (19) combined with label-free quantitative tandem MS (LTQ-Orbitrap). Sequential extraction of 3-day postnatal (P3) mouse epiphyseal cartilage and 3-week neocartilage cultures revealed a marked transition from a high proportion of readily soluble components in P3 extracts to a greater proportion of poorly soluble proteins in neocartilage. Principal component analysis and hierarchical clustering were used to globally assess the inter-relationships between P3 cartilage and neocartilage NaCl and guanidine hydrochloride (GdnHCl) extracts. At a p value cutoff of 0.05, 403 proteins were classified as extract-specific, whereas 125 proteins were classified as tissue sample-specific. Many of the proteins significantly enriched in neocartilage were annotated by the terms cell adhesion, extracellular matrix, and cytoskeletal remodeling. Further statistical analysis identified a third important protein category in which protein solubility was altered between the P3 and neocartilage. Identification of proteins involved in neocartilage maturation has generated novel insight into the fundamental process of cartilage matrix development with potential for further analysis of engineered cartilaginous tissues with biomedical applications.