Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Nucleotide sequence of two 3′-termini of rRNA in the sea urchin Lytechinus variegatus

The 3′-terminus of 26 S rRNA from the sea urchin Lytechinus variegatus has been determined by oligonucleotide fingerprinting, S1 nuclease mapping and terminal nucleotide analysis. There are two species of 26 S rRNA of approximately equal abundance, one 19 nucleotides longer than the other.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

Assignment of 2′-O-Methyltransferases to Modification Sites on the Mammalian Mitochondrial Large Subunit 16 S Ribosomal RNA (rRNA)

Advances in proteomics and large scale studies of potential mitochondrial proteins have led to the identification of many novel mitochondrial proteins in need of further characterization. Among these novel proteins are three mammalian rRNA methyltransferase family members RNMTL1, MRM1, and MRM2. MRM1 and MRM2 have bacterial and yeast homologs, whereas RNMTL1 appears to have evolved later in higher eukaryotes. We recently confirmed the localization of the three proteins to mitochondria, specifically in the vicinity of mtDNA nucleoids. In this study, we took advantage of the ability of 2′-O-ribose modification to block site-specific cleavage of RNA by DNAzymes to show that MRM1, MRM2, and RNMTL1 are responsible for modification of human large subunit rRNA at residues G¹¹⁴⁵, U¹³⁶⁹, and G¹³⁷⁰, respectively.     

Tethering of Poly(A)-binding Protein Interferes with Non-translated mRNA Decay from the 5′ End in Yeast

The decay of eukaryotic mRNA is triggered mainly by deadenylation, which leads to decapping and degradation from the 5′ end of an mRNA. Poly(A)-binding protein has been proposed to inhibit the decapping process and to stabilize mRNA by blocking the recruitment of mRNA to the P-bodies where mRNA degradation takes place after stimulation of translation initiation. In contrast, several lines of evidence show that poly(A)-binding protein (Pab1p) has distinct functions in mRNA decay and translation in yeast. To address the translation-independent function of Pab1p in inhibition of decapping, we examined the contribution of Pab1p to the stability of non-translated mRNAs, an AUG codon-less mRNA or an mRNA containing a stable stem-loop structure at the 5′-UTR. Tethering of Pab1p stabilized non-translated mRNAs, and this stabilization did not require either the eIF4G-interacting domain of Pab1p or the Pab1p-interacting domain of eIF4G. In a ski2Δ mutant in which 3′ to 5′ mRNA degradation activity is defective, stabilization of non-translated mRNAs by the tethering of Pab1p lacking an eIF4G-interacting domain (Pab1–34Cp) requires a cap structure but not a poly(A) tail. In wild type cells, stabilization of non-translated mRNA by tethered Pab1–34Cp results in the accumulation of deadenylated mRNA. These results strongly suggest that tethering of Pab1p may inhibit the decapping reaction after deadenylation, independent of translation. We propose that Pab1p inhibits the decapping reaction in a translation-independent manner in vivo.     

Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.     

Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit?

Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-A-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.     

Analysis of the Gut Microbiota by High-Throughput Sequencing of the V5–V6 Regions of the 16S rRNA Gene in Donkey

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5–V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.     

The Thermus thermophilus5S rRNA–Protein Complex: Identification of Specific Binding Sites for Proteins L5 and L18 in the 5S rRNA

Three 5S rRNA-binding ribosomal proteins (L5, L18, TL5) of extremely thermophilic bacterium Thermus thermophilushave earlier been isolated. Structural analysis of their complexes with rRNA requires identification of their binding sites in the 5S rRNA. Previously, a TL5-binding site has been identified, a TL5–RNA complex crystallized, and its structure determined to 2.3 Å. The sites for L5 and L18 were characterized, and two corresponding 5S rRNA fragments constructed. Of these, a 34-nt fragment specifically interacted with L5, and a 55-nt fragment interacted with L5, L18, and with both proteins. The 34-nt fragment–L5 complex was crystallized; the crystals are suitable for high-resolution X-ray analysis.     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.