Epsilon Sequence Research Articles

Rosmarinic acid is a novel inhibitor for Hepatitis B virus replication targeting viral epsilon RNA-polymerase interaction.

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the “two phenolic hydroxyl groups at both ends” and the “caffeic acid-like structure” of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.

A Simplified Reconstitution of mRNA-Directed Peptide Synthesis: Activity of the Epsilon Enhancer and an Unnatural Amino Acid

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.

Epsilon as an Initiator of Translation of CAT mRNA in Escherichia coli

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458–466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the ε is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of ε in E. coli cells. Our results show that ε displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine–Dalgarno sequence.

Enhancing Activity of ε in Escherichia coli and Agrobacterium tumefaciens Cells

Epsilon (ε) sequence is a bacterial enhancer of translation found in the bacteriophage T7 gene 10. It is believed that its enhancing effect of ε is due to a base-pairing with the nucleotides 458–467 from the helical domain 17 of Escherichia coli 16S rRNA. To prove this we have taken advantage of the difference of this domain in Agrobacterium tumefaciens and E. coli. To evaluate the significance of nucleotide complementarity for the enhancing activity of ε, a series of nucleotide sequences matching either E. coli or A. tumefaciens domain 17 are cloned in a binary expression vector in front of the chloramphenicol acetyltransferase (CAT) gene. The CAT assay shows that: (i) the ε in combination with an SD consensus sequence increases the yield of CAT in both microorganisms over that obtained with the SD alone; (ii) the ε sequence complementary to the A. tumefaciens domain 17 leads to a 2.71-fold increase in the yield of CAT in homologous cells but not in E. coli cells; (iii) the yield of CAT correlates with the free energy of base-pairing with the helical domain 17 in both microorganisms.

Does the Epsilon Sequence of Phage T7 Function as an Initiator for the Translation of CAT mRNA inEscherichia coli?

Epsilon (ϵ) sequence [UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)] which is naturally occurring at the 5′-untranslated leader of phage T7 gene10mRNA was originally described as a powerful translational enhancer inEscherichia coli.Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability. In this study different sequence derivatives of ϵ were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA inE. coli,but also to function as an independent initiator of translation. It was observed that the ϵ sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA. The natural ϵ sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA. However, when the complementarity of ϵ to 16S rRNA was increased from 9 to 16 nucleotides, ϵ alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence.

Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression

The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.

Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.

Hepadnaviruses, as well as other pararetroviruses, express their pol (P) gene product unfused to the preceding core gene implying that these retroelements have developed a mechanism for initiating assembly and replication that is principally different from the one used by retroviruses and retrotransposons. We have analysed this mechanism for the human hepatitis B virus by using a newly developed, highly sensitive detection method based upon radiolabelling of the P protein at newly introduced target sites for protein kinase A. The results obtained demonstrate that polymerase encapsidation depends on the concomittant encapsidation of the HBV RNA pregenome and that packaging of the viral RNA, in turn, depends on the presence of P protein. Loss of P protein encapsidation by mutations inactivating the HBV RNA encapsidation signal epsilon could be compensated by trans-complementation with recombinant RNA molecules carrying the epsilon sequence. Thus, in contrast to retroviral replication, the interaction of the hepadnaviral P protein and the RNA genome at its packaging signal appears to be crucial for initiating the formation of replication-competent nucleocapsids. Furthermore, RNA control of P protein packaging stringently limits the number of polymerase molecules that can be encapsidated.

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.

A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA.

The selective encapsidation of the hepadnaviral RNA pregenome from an excess of other viral and cellular mRNAs predicts specific protein-RNA interactions involving one or several sites on the pregenome. Using deletion analysis in a transient expression/packaging system in which all relevant viral proteins are provided in trans from a packaging-deficient helper genome, we identified near the 5'-end of the pregenome a 137 nucleotide sequence that is necessary and sufficient for RNA encapsidation; other parts of the 3 kb pregenome were found not to contribute to this process. The encapsidation sequence, which we call epsilon, possesses several interesting features with implications for the pregenome's function in RNA packaging, RNA translation and reverse transcription. (i) epsilon contains several indirect repeat sequences, suggesting a high degree of secondary structure, (ii) epsilon overlaps with the start signal for core gene translation, suggesting a mechanism to regulate the alternative use of the pregenome as core mRNA, (iii) epsilon does not contain the direct repeat sequences known to be involved in the initiation of viral DNA synthesis. Finally, our deletion analysis suggests that ribosomes translating the epsilon sequence from the precore start codon may interfere with genomic RNA packaging.

Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor.

In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor.

In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

Conserved organization of the murine immunoglobulin epsilon gene region: restriction endonuclease maps and switch-region nucleotide sequence.

Various inbred mouse strains showed remarkable conservation of organization in the epsilon germline region as determined by restriction mapping. Restriction fragment length polymorphisms (RFLP) were seen in the epsilon gene region for only two of eight restriction enzymes tested. Furthermore, the RFLP did not correlate with the IgE-response phenotype for the murine strains SJL, SJA, C57BL/6, BALB/c, A/ST, and A/J. The IgE class-switch region (S epsilon) DNA from an SJL genomic clone was sequenced and was compared with S epsilon sequences from BALB/c mice. These S epsilon sequences were at least 95% homologous. Most of the S epsilon sequence differences observed between the two strains were single base pair substitutions, deletions, or insertions. The largest difference between the S epsilon sequences resulted from an insertion of seven contiguous bases seen in the SJL S epsilon region.

Tectonic cycles and sedimentary sequences in the Brazilian intracratonic basins

The geometry and petrology of sedimentary rocks preserved in the three intracratonic basins of the Brazilian craton (Parana, Parnaiba, and Amazon) indicate the history and character of vertical movements of the cratonic area. Cyclic successions of erosional and depositional events are synchronous on the Brazilian craton and are correlated with cratons of other continents. The principal evolutionary stages of the Brazilian craton are interpreted as tectonic-sedimentary cycles, each represented by a stratigraphic record identified as a sequence , and each distinguished by its own special characteristics. Cambrian-Ordovician sedimentary rocks, representing deposition in paraplatform basins during a transitional stage at the close of the Brazilian orogenic cycle, are assigned to the Alpha sequence. The Beta (Ordovician and Silurian), Gamma (Devonian–early Carboniferous), and Delta (late Carboniferous–Late Permian sequences) and the Delta-A subsequence (Middle Triassic–Jurassic), corresponding to geotectonic cycles of the cratonic stabilization stage, indicate accumulation in large subsiding basins. The succession of facies in each of these sequences documents cyclical changes in the ratio of sediment supply to subsidence. Marine transgression and basin subsidence increased progressively through the Devonian phase of the Gamma sequence and declined thereafter, in a pattern similar to that of the North American craton and the Russian platform. The frequency of stratigraphic discordances in the three lower sequences suggests a higher degree of cratonic upwarping, progressively diminishing to a stage of maximum stabilization during Delta-A deposition, perhaps related to conditions immediately prior to rupture of the Gondwana plate. Breakup of the Gondwana plate, accompanied by volcanism and remobilization of cratonic areas, initiated the reactivation stage during which two geotectonic cycles are recognized. The first, represented by the Epsilon sequence (Cretaceous), began with local subsidence in isolated basins, followed by widespread continental sedimentation under platform conditions. The second reactivation cycle was responsible for accumulation of the Zeta sequence (Cenozoic), which was characterized by thin residual deposits on an extensive Tertiary peneplain and by accumulations in Quaternary basins, the latter of minor importance except where adjacent to the uplifted eastern continental margin.