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RNase E isolated from Escherichia coli is contained in a multicomponent \"degradosome\" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3′-to-5′ exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

Recently, we found that a multicomponent ribonucleolytic degradosome complex formed around RNase E, a key mRNA-degrading and 9S RNA-processing enzyme, contains RNA in addition to its protein components. Herein we show that the RNA found in the degradosome consists primarily of rRNA fragments that have a range of distinctive sizes. We further show that rRNA degradation is carried out in the degradosome by RNase E cleavage of A+U-rich single-stranded regions of mature 16S and 23S rRNAs. The 5S rRNA, which is known to be generated by RNase E processing of the 9S precursor, was also identified in the degradosome, but tRNAs, which are not cleaved by RNase E in vitro, were absent. Our results, which provide evidence that decay of mature rRNAs occurs in growing Escherichia coli cells in the RNA degradosome, implicate RNase E in degradosome-mediated decay.

Peri-implantitis is a severe biofilm-associated infection affecting millions worldwide. This cross-sectional study aimed to identify taxonomic and functional biomarkers that reliably indicate peri-implantitis by utilizing paired data from full length 16S rRNA gene amplicon sequencing (full-16S) and metatranscriptomics (RNAseq) in 48 biofilm samples from 32 patients. Both full-16S and RNAseq analyses revealed significant differences between healthy and peri-implantitis samples, with a shift toward anaerobic Gram-negative bacteria in peri-implantitis. Metatranscriptomics identified enzymatic activities and metabolic pathways associated with peri-implantitis and uncovered complex peri-implant biofilm ecology related to amino acid metabolism. Integrating taxonomic and functional data enhanced predictive accuracy (AUC = 0.85) and revealed diagnostic biomarkers including health-associated Streptococcus and Rothia species and peri-implantitis-associated enzymes (urocanate hydratase, tripeptide aminopeptidase, NADH:ubiquinone reductase, phosphoenolpyruvate carboxykinase and polyribonucleotide nucleotidyltransferase). Thus, biofilm profiling at taxonomic and functional levels provides highly predictive disease biomarkers, laying the foundation for novel diagnostic and personalized treatment approaches for peri-implant disease.

Background During the early stages of seed development many genes are under dynamic regulation to ensure the proper differentiation and establishment of the tissue that will constitute the mature grain. To investigate how miRNA regulation contributes to this process in barley, a combination of small RNA and mRNA degradome analyses were used to identify miRNAs and their targets. Results Our analysis identified 84 known miRNAs and 7 new miRNAs together with 96 putative miRNA target genes regulated through a slicing mechanism in grain tissues during the first 15 days post anthesis. We also identified many potential miRNAs including several belonging to known miRNA families. Our data gave us evidence for an increase in miRNA-mediated regulation during the transition between pre-storage and storage phases. Potential miRNA targets were found in various signalling pathways including components of four phytohormone pathways (ABA, GA, auxin, ethylene) and the defence response to powdery mildew infection. Among the putative miRNA targets we identified were two essential genes controlling the GA response, a GA3oxidase1 and a homolog of the receptor GID1 , and a homolog of the ACC oxidase which catalyses the last step of ethylene biosynthesis. We found that two MLA genes are potentially miRNA regulated, establishing a direct link between miRNAs and the R gene response. Conclusion Our dataset provides a useful source of information on miRNA regulation during the early development of cereal grains and our analysis suggests that miRNAs contribute to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.

Abstract The gene encoding for polynucleotide phosphorylase (pnp) of a new biovar of Staphylococcus aureus subsp. aureus (NBSA) has been isolated from a genomic library of strain M280(0). The coding region consisted of a 1094-bp HindIII–HindIII DNA fragment encoding for a protein of 277 amino acids with a calculated molecular mass of 29.5 kDa. The nucleotide sequence of the structural gene, contained a continuous open reading frame of 836 bp, showed significant homology with the genes of bacterial polynucleotide phosphorylase from Bacillus subtilis (67.7% identity), from Haemophilus influenzae (62.47% identity), from Pseudomonas luminescens (61.6% identity), and from Escherichia coli (59.7% identity). DNA–DNA and DNA–colony slot-blot hybridizations demonstrated that the pnp gene, employed as a molecular probe, is specific for the identification of NBSA strains.

Final trimming of the 3′ terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism. However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally. Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate. Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH. Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing. Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity. RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates. The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.

An enzyme, purified 300-fold from Escherichia coli infected with bacteriophage T4, catalyzes the conversion of 5′-termini of polyribonucleotides to internal phosphodiester bonds. The reaction requires ATP and Mg++. For every 5′-32P terminus rendered resistant to alkaline phosphatase, an equal amount of AMP and PPi are formed. Various polyribonucleotides are substrates in the reaction; to date, the best substrate is [5′-32P]polyriboadenylate. With the latter substrate, no evidence of intermolecular reaction was obtained. However, the 5′-32P termini of poly(A) rendered resistant to alkaline phosphatase are also resistant to attack by RNase II, polynucleotide phosphorylase, and low concentrations of venom phosphodiesterase. Since the product formed with poly(A) lacks 3′-hydroxyl ends, as measured with these exonucleases, the enzyme appears to convert linear molecules of polyriboadenylate to a circular form by the intramolecular covalent linkage of the 5′-phosphate end to the 3′-hydroxyl terminus.

The question of whether or not a cellular ribonuclease is involved in the cleavage of 16S ribosomal RNA by colicin E3 was investigated. For this purpose ribosomes from strains devoid of some ribonucleases or ribosomes in which ribonucleases had been inactivated by heat or removed by extensive washings were used for the colicin reaction. Since the 16S RNA of all these different ribosomes, and even of the most extensively washed ribosomes, was cleaved by colicin E3, it is suggested that cellular ribonucleases are not involved in colicin E3 action. Thus, colicin E3 seems to be a unique endoribonuclease.

The DNA polymerase of Rauscher murine leukemia virus is strongly and specifically inhibited by nontemplate, single-stranded polyribonucleotides with either the resident viral RNA, native calf-thymus DNA, or poly[d(A-T)] as templates. These inhibitory homo-polymers are apparently bound to the template site of the polymerase, since they interact competitively with the template. The strength of the inhibition depends on the particular homopolymer used: poly(U) > poly(G) ≫ poly(A) > poly(C). The Kifor poly(U) was 0.08 μ g/ml, which represents an apparent affinity six times greater than that observed for viral RNA. No such inhibition was observed with a highly purified DNA polymerase from mouse embryos or the Escherichia coli enzyme.