Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.     

Ribosomal frameshifting in yeast viruses

Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting.     

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three-dimensional reconstruction

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al ., Nature , 376, 441–444, 1995; Stark et al ., Structure , 3, 815–821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.     

A Recombinant Collagen–mRNA Platform for Controllable Protein Synthesis

We have developed a collagen–mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin‐healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell‐free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase–mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.     

The Proto-Ribosome: An Ancient Nano-machine for Peptide Bond Formation

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A- to P-site passage of the tRNA 3′ terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement, and guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate-mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. Since in the contemporary ribosome, the symmetry relates only to the backbone fold and nucleotide orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication.     

In situ hybridization and immunofluorescence on resin‐embedded tissue to identify the components of Nissl substance

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30‐μm cryostat and 1–3‐μm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin‐labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin‐embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30‐μm cryostat sections, the most striking feature of ISH in the thinner resin (2–3 μm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Ribosomal Protein S5e is Implicated in Translation Initiation through its Interaction with the N‐Terminal Domain of Initiation Factor eIF2α

A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met‐tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde‐induced protein–protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, “e” stands for “eukaryotic”) and eIFs within the mammalian PIC, assembled on either model canonical or IRES‐containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N‐terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start‐codon selection by the PIC.     

Image-EELS for in situ estimation of the phosphorus content of RNP granules

Electron energy-loss spectroscopic imaging is performed on ultrathin sections, in order to approach the in-situ distribution of individual elements in the cellular constituents. In a previous ultrastructural study of salivary gland cells of Chironomus thummi and Ch. tentans by means of electron spectroscopic imaging and cytochemistry, we have described a new type of phosphorus-rich small granular ribonucleoprotein (RNP) component in the nucleoplasm of these cells; an attempt is made in the present work to estimate the phosphorus content of this component by the above mentioned method, using ribosomes as internal standard.   Results show that these RNP granules contain, on average 3600 phosphorus atoms per granule. Another nuclear constituent also studied, the Balbiani ring granule, contains, on average, 43 000 phosphorus atoms per granule.     

Saccharomyces cerevisiae,a Powerful Model for Studying rRNA Modifications and Their Effects on Translation Fidelity

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.