ULTRASTRUCTURE OF THE NUCLEOLUS AND REPLICATION SITES OF RIBOSOMAL DNA IN Allium cepa

Our conventional EM observations indicate that the nucleolus of Allium cepa is composedof the fibrillar centre (FC), fibrillar component (F) and granular region (G). FC is an elec-tron-lucid zone which contains condensed or loosened chromatin. F is a circular, highly elec-tron-dense component which surrounds FC and consists of compact fibrils. EM autoradiographic(EM ARG) studies with ~3H- UdR reveal that the synthesis of ribosomal RNA (rRNA) takesplace in F. G is situated around F and composed of granules about 25 to 30 nm in diameter.EM ARG studies with ~3H- TdR demonstrate that silver grains are predominantly located in Gof the nucleolar periphery and the region of the nucleolus-associated chromatin. In addition,~3H- TdR labels are also present, but with a much lower frequency, in FC. F and G of the nu-cleolar interior. According to the distribution and sphere of silver grains in different compo-nents of the nucleolus, we suggest that the replication sites of ribosomal DNA (rDNA) aremainly located in     

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m2G966 and m?C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low μM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.     

Protein factors mediating selenoprotein synthesis

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.     

Ribosome Subunit Stapling for Orthogonal Translation in E. coli

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl‐transferase center, for unnatural polymer synthesis in cells.     

Histidine affinity chromatography-based methodology for the simultaneous isolation of Escherichia coli small and ribosomal RNA

Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.     

Nonribosomal peptide synthesis in animals: the cyclodipeptide synthase of Nematostella

Cyclodipeptide synthases (CDPSs) are small enzymes structurally related to class-I aminoacyl-tRNA synthetases (aaRSs). They divert aminoacylated tRNAs from their canonical role in ribosomal protein synthesis, for cyclodipeptide formation. All the CDPSs experimentally characterized to date are?bacterial. We show here that a predicted CDPS from the sea anemone Nematostella vectensis is an active CDPS catalyzing the formation of various cyclodipeptides, preferentially containing tryptophan. Our findings demonstrate that eukaryotes encode active CDPSs and suggest that all CDPSs have?a similar aminoacyl-tRNA synthetase-like architecture and ping-pong mechanism. They also raise questions about the biological roles of the cyclodipeptides produced in bacteria and eukaryotes.     

8-METHOXYPSORALEN DNA INTERSTRAND CROSS-LINKING OF THE RIBOSOMAL RNA GENES IN Tetrahymena thermophila. DISTRIBUTION, REPAIR AND EFFECT ON rRNA SYNTHESIS

Abstract— The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA gencs (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of ≤ 1/2 × 10⁴ base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis ( Bam HI and Cla I). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is suficient to block KNA synthesis. Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive.     

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with ^tzG and ^thG, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs’ substrate recognition features.     

Structure and function of the acidic ribosomal stalk proteins

The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The physiological role of the other dimerization mode is unclear and is in apparent contradiction to distances measured by fluorescence resonance energy transfer. The discrepancies between the crystal structures and results from other physico-chemical methods may partly be a consequence of the dynamic functions of the proteins, necessitating a high flexibility.     

The 5-aminolaevulinic acid-based photodynamic effects on nuclei and nucleoli of HL-60 leukemic granulocytic precursors

To provide more information on the 5-aminolaevulinic acid (ALA)-based photodynamic effect (PDE) on nuclei and nucleoli of individual leukemic cells, these structures were studied in cultured HL-60 cells which originated from leukemic highly immature and less differentiated precursors of granulocytes. The nuclear morphology was visualized by panoptic May-Grünwald/Giemsa staining and cytochemical method for DNA, nucleoli were visualized by cytochemical methods for the demonstration of RNA and silver stainable proteins including those of interphase silver stained nucleolus organizer regions (AgNORs). In most cells ALA-based photodynamic treatment (PDT) produced marked alterations such as formation of apoptotic bodies, and large condensation of nuclear chromatin structure but without nuclear segmentation. Such changes are in harmony with the apoptotic process induced in these cells but without previous terminal differentiation. In nucleoli ALA-based PDT produced the reduction and disappearance of nucleolar silver stainable particles (SSPs) representing AgNORs which apparently reflected the alteration of the nucleolar biosynthetic activity and cell proliferation. The latter is also reflected by the disappearance of mitotic divisions. On the other hand, a small subpopulation of cells was less sensitive or resistant to the ALA-based PDE since they did not show mentioned nuclear and nucleolar alterations.     

Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond

Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds.     

Morphological studies of deformed polybutylene terephthalate (PBT)

PBT is a semi-crystalline thermoplastic polymer whose deformation behavior highly depends on processing parameters. This makes it a model polymer for investigating morphological changes caused by deformation on the spherulitic and lamellar level. In the neck region all states of deformation of the spherulites are observed. Even in the fibrillar phase the borders of the spherulites remain visible. The spherulitic structure is not totally destructed in the neck. The lamellar structure of the fibrillar phase significantly differs from that of the spherulitic region. The lamellae are orientated with respect to the direction of deformation and the lamellae heights are reduced distinctly. Scanning electron microscopy of fracture surfaces reveals for some samples a sharp frontier between spherulitic and fibrillar region. This leads to the conclusion that the necking process may be a phase transition between an isotropic and a highly orientated phase, as predicted for a Van der Waals network.     

Structure-function Relationships in Human Hypoxanthine-guanine Phosphoribosyltransferase （HGPRT） by Random Mutagenesis

Introduction Hypoxanthine guaninephosphoribosyltransferase(HGPRT,EC2.4.2.8)isakeyenzymeofthepurine salvagepathway,whichallowsrecyclingofpurinebases intoDNAandRNA.Itiswidelydistributedinnature andhasbeenstudiedbothinprokaryotesandeu karyotes.Inhumans,acomp…