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PubMed1983

Regulatory steps in the initiation of protein synthesis.

Regulation of protein synthesis is being exerted at different levels with a different extent of attenuation. The major control module seems to work by the inactivation of the elF-2 recycling which enables the cell to shift down from a high rate of initiation to a low rate. Certain events in the cell cycle like mitosis do show such a drastic change in initiation rate. It is suggested that modifications of elF-2 by phosphorylation of the alpha-subunit by different protein-kinases is the basis for such a control mechanism. Already two protein kinases of this type have been described, the hemin-regulated inhibitor and the ds-RNA activated inhibitor from interferon-treated cells. On the other hand modifications of the beta-subunit by other metabolic events, for instance low NADH/NAD+ ratio, can as yet not be excluded. Other conditions like amino acid starvation, serum deprivation, heat-shock and virus-infection seem to evoke quite different strategies. In some cases it has been demonstrated that inactivation of mRNA binding factors as elF-4B and elF-4E, favour the translation of low-dependence, i.e. low secondary structure, messengers. It shall be worthwhile to establish whether the mRNA's with such low degree of secondary structure encoded proteins that are aimed at the survival of the cell under extreme metabolic or stress conditions. Much more work on the structure and nucleotide sequences of the leader sequence is needed to prove these hypothetical points.

Kinetics↗
PubMed1999

Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Oligo-2'-fluoro-2'-deoxynucleotide N3'-->P5' phosphoramidates: synthesis and properties.

Uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 2'-fluoro-2'-deoxy-pyrimidine nucleosides were synthesized using an efficient interphase amidite transfer reaction. The 3'-amino group of solid phase-supported 2'-fluoro-2'-deoxynucleoside was used as an acceptor and 5'-diisopropylamino phosphoramidite as a donor of a phosphoramidite group in the tetrazole-catalyzed exchange reaction. Subsequent oxidation with aqueous iodine resulted in formation of an internucleoside phosphoramidate diester. The prepared oligo-2'-fluoro-nucleotide N3'-->P5' phosphoramidates form extremely stable duplexes with complementary nucleic acids: relative to isosequential phosphodiester oligomers, the melting temperature Tm of their duplexes with DNA or RNA was increased approximately 4 or 5 degrees C per modification respectively. Moreover, these compounds are highly resistant to enzymatic hydrolysis by snake venom phosphodiesterase and they are 4-5 times more stable in acidic media (pH 2.2-5.3) than the parent oligo-2'-deoxynucleotide N3'-->P5' phosphoramidates. The described properties of the oligo-2'-fluoronucleotide N3'-->P5' phosphoramidates suggest that they may have good potential for diagnostic and antisense therapeutic applications.

Base Sequence↗
PubMed1984

Biosynthesis of rainbow trout (Salmo gairdnerii) hemoglobins in vitro.

Cell free systems were established to analyze the biosynthesis of trout hemoglobins I, II and III. HbI, II and III were synthesized in trout erythroid cell lysates as well as in a mRNA dependent rabbit reticulocyte lysate supplemented with trout erythroid cell polyA-RNA. The newly synthesized hemoglobins apparently contained the N alpha-acetyl modification at their alpha-chain amino terminals since they co-migrated with carrier trout hemoglobins that are known to contain the modification. This observation suggests that the acetylation is determined by the information encoded in the mRNA.

Anemia↗
PubMed2004

Evolution of a T7 RNA polymerase variant that transcribes 2'-O-methyl RNA.

Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior. In particular, modifications at the 2'-OH of ribose are important for enhancing the stability of RNA. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2' position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript.

DNA-Directed RNA Polymerases↗
PubMed1982

The tRNA-like structure at the 3' terminus of turnip yellow mosaic virus RNA. Differences and similarities with canonical tRNA.

The 3' terminus of TYMV RNA, which possesses tRNA-like properties, has been studied. A 3' terminal fragment of 112 nucleotides was obtained by cleavage with RNase H after hybridization of a synthetic oligodeoxynucleotide to the viral RNA. The accessibility of cytidine and adenosine residues was probed with chemical modification. Enzymatic digestion studies were performed with RNase T1, nuclease S1 and the double-strand specific RNase from the venom of the cobra Naja naja oxiana. A model is proposed for the secondary structure of the 3' terminal region of TYMV RNA comprising 86 nucleotides. The main feature of this secondary structure is the absence of a conventional acceptor stem as present in canonical tRNA. However, the terminal 42 nucleotides can be folded in a tertiary structure which bears strong resemblance with the acceptor arm of canonical tRNA. Comparison of this region of TYMV RNA with that of other RNAs from both the tymovirus group and the tobamovirus group gives support to our proposal for such a three-dimensional arrangement. The consequences for the recognition by TYMV RNA of tRNA-specific enzymes is discussed.

Base Sequence↗
PubMed2005

Introduction of hypermodified nucleotides in RNA.

The anticodon domain of lysine transfer ribonucleic acid (tRNA) is a model system for investigation of the structural and biochemical effects of nucleoside posttranscriptional modification. To enable detailed study of the biophysical and structural effects of hypermodified nucleosides, methods have been developed to synthesize RNA oligonucleotides containing the modified nucleosides found in lysine tRNA. We describe in detail the synthesis of protected phosphoramidites of the nucleosides methylaminomethyl-2-thiouridine (mnm5s2U), methylcarboxymethyl-2-thiouridine (mcm5s2U), and 2-thiomethyl-N-6-carbamoylthreonyl-adenosine (ms2t6A). We also describe methods for using these nucleoside phosphoramidite reagents to synthesize RNA oligonucleotides with modified nucleosides incorporated at the specific sequence locations corresponding to their positions in the native lysine tRNAs.

Nucleotides↗
PubMed1995

Codon recognition by tRNA molecules with a modified or unmodified uridine at the first position of the anticodon.

Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis. We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon. One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U). These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated. The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon.

Anticodon↗
PubMed1993

Identification of specific contacts in T3 RNA polymerase-promoter interactions: kinetic analysis using small synthetic promoters.

The T7, T3, and SP6 RNA polymerases recognize very similar, yet distinct, promoter sequences. The high homology among the promoter sequences suggests that differential promoter recognition must derive from relatively small changes in the protein. Steady-state kinetic analyses of transcription from the T3 consensus promoter and from promoters modified in the region critical to specific recognition reveal details concerning which functional groups contribute to this recognition. Modifications include base pair substitutions, single base substitutions (mismatches), and simple functional group modifications at unique sites in the promoter. The results show that T3 RNA polymerase recognizes the amino group on the nontemplate cytidine in the major groove at position -10, while the identity of the base on the template strand is less critical to binding. In contrast, recognition at position -11 allows a greater range of modifications and seems to have a more complex recognition. The results do not seem to be consistent with a single recognition contact at this position; however, some groups may be ruled out as simple recognition contacts. While major groove modifications weaken binding at positions -10 and -11, the removal of an exocyclic amino group from the minor groove at either position does not disrupt binding, further supporting a model for promoter recognition in which the enzyme binds to one face of closed duplex DNA in this region. The effects of these changes in the DNA structure on the kinetics of initiation are compared to complementary results from the T7 system.

Bacteriophage T7↗
PubMed2004

[Advances in research on lignin biosynthesis and its genetic engineering].

Lignin, one of the main components in vascular plants, is important for the adaptation of terrestrial plants to environment during evolution. However, its presence in plants has negative effects on wood processing during pulping and stock breeding. Therefore much attention has been focused on the regulation of lignin biosynthesis. The pathways leading to the synthesis of lignin polymers have been studied for decades. Much understanding of lignin biosynthesis has been advanced. This paper reviewed the recent progress made in the various steps associated with monolignol biosynthesis. It includes the catalysis by three enzymes, i.e. p-coumarate-3-hydroxylase (C3H), ferulate-5-hydroxylase (F5H) and caffeic acid 3-O-methyltransferase (COMT); the multiform biosynthetic pathway of syringyl (S) lignin in angiosperms; the biosynthesis route of guaiacyl (G) and syringyl (S) lignin specifically regulated by cinnamyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD) and the formation of the lignin macromolecule. Based on the elucidation of lignin biosynthesis pathway, it has also been given the achievements in lignin gene engineering. Many studies were concentrated on the modification of lignin content and composition. In some cases, the potential value of transgenic plants with modified lignin beneficial for pulping has been demonstrated. To better understand the mechanism of lignin biosynthesis and improve the properties of plants, new biotechnological strategies can be developed, which include combinatorial modification of multiple lignin traits in plants through multigene cotransformation, transcriptional control of lignin biosynthesis and the application of RNA interference. The identification of novel genes by molecular and genetic approaches will be useful in opening up new avenues of lignin modification in the future.

Genetic Engineering↗
PubMed1991

Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs.

To investigate whether RNA editing in plant mitochondria modifies structural RNAs as well as protein-coding RNAs we compared the genomic-encoded information with the respective transcripts of several genes in Oenothera. The genes analysed are the 5S, 18S and 26 S rRNAs, the alpha-subunit of ATPase (atpA), cytochrome b (cytb), orfB, which is located upstream of cytochrome oxidase subunit III, and the respective leader, trailer and spacer sequences. All open reading frames were found to be edited to some degree. The atpA coding region has the least edited mRNA in Oenothera mitochondria, with only four nucleotides altered in the 1533 nucleotide open reading frame. From this analysis we conclude that frequent RNA editing is indicative of functional protein coding regions in plant mitochondria. The extensive editing in orfB, for example, suggests that this orf codes for a mitochondrial protein. No RNA editing event was found in the 5S rRNA or in the 1824 nucleotides analysed of the 18S rRNA, but two nucleotides were found to be altered in the 1970 nucleotides compared for the 26S rRNA. One nucleotide alteration has changed C to U, the other in reverse U to C. However, only one of five cDNA clones covering this region shows the modifications, similar to many silent editing events in open reading frames. RNA editing in the structural RNAs thus does not seem to be essential for their function in the mitochondrial ribosome.

Adenosine Triphosphatases↗
PubMed2004

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe.

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.

Adenine↗
PubMed2005

A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503.

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.

Anti-Bacterial Agents↗
PubMed1997

Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids.

In this study we report for the first time the binding affinity of RNase H1 for oligonucleotide duplexes. We used a previously described 17-mer antisense sequence [Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., & Freier, S. M. (1992) J. Biol. Chem. 267, 19954-19962] hybridized to a complementary oligoribonucleotide to evaluate both the binding affinity and the catalytic rate of RNase H1. The dissociation constants (Kd) of RNase H1 for the various substrates tested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes. Catalytic rates were determined using heteroduplex substrates containing chimeric antisense oligonucleotides composed of a five-base deoxynucleotide sequence flanked on either side by chemically modified nucleotides. We find that the enzyme preferentially binds A-form duplexes: RNase H bound A-form duplexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-strand oligonucleotides. The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes containing various 2'-sugar modifications, while the cleavage rates for these chemically modified substrates were without exception slower than for the wild type substrate. The introduction of a single positively charged 2'-propoxyamine modification into the chimeric antisense oligonucleotide portion of the heteroduplex substrate resulted in both decreased binding affinity and a slower rate of catalysis by RNase H. The cleavage rates for heteroduplexes containing single-base mismatch sequences within the chimeric oligonucleotide portion varied depending on the position of the mismatch but had no effect on the binding affinity of the enzyme. These results offer further insights into the physical binding properties of the RNase H-substrate interaction as well as the design of effective antisense oligonucleotides.

Base Sequence↗
PubMed1998

Structural and dynamic helix geometry alterations induced by mismatch base pairs in double-helical RNA.

A ribooligonucleotide microhelix derived from the acceptor stem of Escherichia coli tRNA(Ala) having a C3-A70 mismatch in place of the G3-U70 wobble pair in the wild-type tRNA(Ala), and a sequence variant with a regular U3-A70 base pair have been investigated by NMR. In vivo, suppressor tRNA(Ala) variants with C3-A70 (as well as several other) mismatch pairs are substrates for alanyl-tRNA synthetase (ARS), supporting the hypothesis of an 'indirect' recognition of the identity element 3-70 mismatch pair via structural modifications caused by the mispair in comparison to canonical A-RNA helices. It is demonstrated that the C-A mismatch likewise induces helix geometry alterations, in particular with respect to base stacking in the vicinity of the mismatch. However, with reference to the 'wild-type' G3-U70 microhelix, destacking in the C3-A70 acceptor stem duplex occurs in the opposite direction from the mismatch pair. Therefore it is concluded that the locally enhanced conformational flexibility or dynamics associated with the structural changes induced by the mismatch pairs could be an essential prerequisite for optimal adaptation of the tRNA(Ala) acceptor stem to the contact region of the ARS.

Escherichia coli↗
PubMed1995

NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures.

Software packages NUPARM and NUCGEN, are described, which can be used to understand sequence directed structural variations in nucleic acids, by analysis and generation of non-uniform structures. A set of local inter basepair parameters (viz. tilt, roll, twist, shift, slide and rise) have been defined, which use geometry and coordinates of two successive basepairs only and can be used to generate polymeric structures with varying geometries for each of the 16 possible dinucleotide steps. Intra basepair parameters, propeller, buckle, opening and the C6...C8 distance can also be varied, if required, while the sugar phosphate backbone atoms are fixed in some standard conformation in each of the nucleotides. NUPARM can be used to analyse both DNA and RNA structures, with single as well as double stranded helices. The NUCGEN software generates double helical models with the backbone fixed in B-form DNA, but with appropriate modifications in the input data, it can also generate A-form DNA and RNA duplex structures.

Base Composition↗
PubMed2006

Structure and function of protein modules in chromatin biology.

Chromatin-mediated gene transcription or silencing is a dynamic process in which binding of various proteins or protein complexes can displace nucleosomal histones from DNA to relieve repression or drive the gene into a highly repressed, silent state. Covalent modifications to DNA and histones associated with chromatin structural change play a crucial role in transcriptional regulation, with particular modifications on certain residues associated with a specific transcriptional outcome. In recent years a number of structural domains have been identified within chromatin-associated proteins, including DNA or RNA binding domains, protein-protein interaction domains and domains that recognize specific covalent modifications to histone tails. In this review we discuss the structural features of these protein modules and the functional roles they play in chromatin biology.

Acetylation↗
PubMed2005

5-Azacytidine suppresses RNA polymerase II recruitment to the SLPI gene.

Histone methylation is regarded as a stable modification important in the epigenetic regulation of gene expression. Transcriptionally active chromatin is methylated at H3-K4 whereas repressed chromatin is methylated at H3-K9. To investigate the role of histone methylation in an acute inflammatory response, A549 cells were treated with IL-1beta and/or the methylase inhibitor 5-azacytidine (5-aza), and histone H3-K4 methylation levels and transcription of secretory leukocyte protease inhibitor (SLPI) were measured. IL-1beta stimulation enhanced histone H3-K4 tri-methylation across the SLPI coding region at 24h. In parallel, IL-1beta enhanced recruitment of RNA polymerase II to the SLPI gene. 5-aza attenuated both H3-K4 tri-methylation and RNA polymerase II recruitment to a similar extent resulting in reduced SLPI mRNA and protein levels. These data suggest that in addition to epigenetic regulation of constitutive SLPI expression, H3-K4 tri-methylation may play a role in stimulated SLPI expression by modulating RNA polymerase II recruitment and subsequent gene transcription.

Animals↗