Can initiate synthesis de novo ..
Flock house virus RNA polymerase initiates RNA synthesis de novo and possesses a ..
DNA-dependent RNA polymerases can also initiate RNA synthesis de novo
The purified EAV nsp9/RdRp was able to initiate RNA synthesis de novo in a template-specific manner, but no activity was found on poly(A) templates and more structured heteropolymeric viral RNA templates. This template specificity may be a mechanism to prevent initiation of RNA synthesis on the polyadenylated structured mRNAs that are abundantly present in the host cell's cytoplasm. Specific initiation of RNA synthesis may be regulated by critical RNA-RNA or protein-RNA interactions involving the viral RNA template, and ongoing studies using the purified EAV nsp9/RdRp aim to identify such determinants of initiation and template specificity. For example, the recently described hairpin near the 3′ terminus of the EAV genome is a likely target for such interactions (). The ability to purify an enzymatically active EAV RdRp will facilitate its further functional and structural characterization and will provide insight into the molecular basis of RNA polymerization. Such detailed molecular knowledge of viral RdRps also may contribute to the identification of selective inhibitors of this important class of viral enzymes.
Several recent studies have experimentally verified that HCV NS5B is an RdRp () that catalyzes ribonucleotide polymerization on heteropolymer as well as homopolymer RNA templates in vitro (, , –, ). In an attempt to further reveal the intrinsic properties of HCV NS5B, cDNA of NS5B was directly amplified from HCV viral RNA extracted from an HCV-infected patient's serum and used to construct recombinant baculoviruses. Recombinant NS5B proteins were subsequently purified from insect cells infected with recombinant baculoviruses, and RdRp activities were examined in vitro. These efforts have resulted in several active NS5B proteins with various levels of RdRp activity (Table ). The identified amino acid differences among different isolates likely account for the inactivity or variable activities of NS5B proteins in catalyzing in vitro RNA synthesis (Table ). However, efforts were not made in this study to determine the effects of the specific amino acid differences on RdRp activity. In addition, nearly half of the NS5B isolates cloned from a patient's serum were found to be inactive or weakly active in the in vitro RdRp assay. The low level of HCV replication in vivo may be associated with a large portion of inactive or weakly active NS5B proteins. These data also show that NS5B proteins with high RdRp activity exist in the virus pool and that they can be identified by examining multiple isolates. The 1b-42 described here possesses the highest RdRp activity among all isolates evaluated. It was therefore purified to >90% homogeneity (Fig. ), and subjected to extensive characterization in a RdRp assay in vitro. Results derived from characterization of 1b-42 NS5B have led to several interesting findings.
RNA polymerase, which can initiate synthesis of an mRNA ..
Divalent metals are known to modulate substrate selectivity of DNA-dependent RNA polymerases (, ). For the HCV RdRp, Mn2+ improved the Km value for the initiation GTP from 103 to 3 μM. Mn2+ also decreased primer extension since its addition to reactions containing Mg2+ not only increased de novo initiation but also reduced primer extension activity.
An emerging theme in studies of viral RdRp structure and function is that metals can regulate the activities of viral RdRps in addition to performing nucleotide polymerization. The dengue virus replicase was suggested to exist in at least two conformational states that are in dynamic equilibrium (). The so-called closed conformer, formed at lower temperatures, is more capable of de novo initiation, while the form that preferentially exists at higher temperatures favors primer-dependent synthesis. The crystal structure of the calicivirus RdRp also exists in closed and open conformations that are, respectively, active and inactive for RNA synthesis in vitro (). The closed conformer of this RdRp contained two Mn2+ ions occupying the active site of the protein. We predict that Mn2+ will prove to have a similar effect on the structure of the HCV and possibly other flaviviral RdRps.
De Novo Initiation of RNA Synthesis by the RNA …
Recombinant RdRps from HCV, GBV, and BVDV were purified to single band in a Coomassie blue-stained gel (Fig. ). Characterization of de novo-initiated RNA synthesis by the HCV and BVDV RdRps has been reported earlier (, , ). The GBV RdRp was demonstrated to initiate RNA synthesis by a de novo mechanism and to extend from a primer (C. T. Ranjith-Kumar, J. Lin-Goerke, L. A. Gutshall, et al, unpublished data).
RNA synthesis assays primarily used a 19-nucleotide (nt) RNA, LE19 (Fig. ), whose sequence is derived from the 3′ end of the minus-strand BVDV genome. There are many reasons for using LE19. First, since it has only one cytidylate at the initiation position, LE19 allows us to use GTP only for initiation and not for elongation. Second, as will be shown in this work, it directs robust levels of RNA synthesis by all three recombinant RdRps. Third, LE19 can be used to detect four RdRp activities within the same reaction: de novo initiation, primer extension, terminal transferase activity (), and RNA template switch leading to the formation of recombinant products (). Terminal transferase activity would add nontemplated nucleotides to LE19, resulting in products longer than 19 nt. De novo initiation from the 3′-terminal cytidylate that terminated at the 5′ end of LE19 would result in a 19-nt newly synthesized RNA. Should the de novo-initiated ternary complex not terminate and use a second template to continue synthesis (as shown in reference ), the product of this template switch event will be ca. 38 nt long. Finally, should two LE19 molecules anneal through six base-paired nucleotides involving the 3′ ends of the RNAs (Fig. , schematic on the right) to form a structure capable of primer extension, the 32-nt primer extension product, based on its sequence, can form in the absence of GTP (Fig. ).
Regulation of De Novo-Initiated RNA Synthesis in ..
initiate RNA synthesis de novo – without a primer
We determined the crystal structures of T7 RNA polymerase complexes captured during the de novo RNA synthesis
Some RNA-dependent RNA polymerases can also initiate RNA ..
19/10/2015 · De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase ..
can synthesize RNA chains de novo ..
DNA, RNA and protein synthesis
The genetic material is stored in the form of DNA in most organisms
There are no data on the structure, function, or in vitro activity of the RdRps of arteriviruses, which are distantly related to those of CoVs (for recent reviews, see references and ). In this study, we report the expression in Escherichia coli and purification of the active EAV nsp9/RdRp and address the mechanism of initiation of arterivirus RNA synthesis. The arterivirus RdRp sequences were compared to those of their CoV homologues and to other RdRps with known initiation mechanisms and three-dimensional structures. In an in vitro assay, the EAV RdRp was able to initiate RNA synthesis on homopolymeric templates using a de novo mechanism. Initiation of RNA synthesis was also studied in vivo by using a reverse genetics approach, which demonstrated the importance of the 3′-terminal nucleotides of the EAV genome. This work provides a basis for further characterization of the arterivirus RdRp and dissection of the molecular basis of RNA polymerization. Such detailed molecular knowledge will be essential for developing therapeutics that selectively inhibit viral RdRps in vivo.
Glossary | Linus Pauling Institute | Oregon State University
Like all other RdRps, nidovirus RdRps contain the conserved catalytic motifs A, B, and C in the palm subdomain, as well as motif F in the finger subdomain, which contacts the incoming nucleotides. They also contain motif E, a structural (not sequence) motif found in both primer-dependent and de novo-initiating RdRps, forming the connection between the palm and thumb domains. Motif G, also present in nidovirus RdRps, is a sequence signature characteristic of primer-dependent RdRps (). Evidence for a primer-dependent in vitro activity of the coronavirus (CoV) RdRp (nsp12) came from a study of the severe acute respiratory syndrome CoV (SARS-CoV) enzyme, which was found to be active on poly(A) templates by using oligo(U) to prime initiation (). In a recent report, a second SARS-CoV RdRp (nsp8) was identified that was able to initiate RNA synthesis de novo with low processivity and low fidelity (). This second RdRp was proposed to act as a primase, possibly generating the primers for the canonical primer-dependent nsp12/RdRp.
DNA Virus Replication - Microbiology Book
In the absence of an exogenous primer, some RNA template can use its 3′-terminal sequence to fold back and form a hairpin structure. The folded back sequence can serve as an endogenous primer to initiate RNA synthesis, and this mechanism is called “copy-back” initiation, which is also primer dependent , , . If such a mechanism is used, we should detect the product with the double size of the RNA template, as the synthesized strand is covalently linked to the template via the hairpin. Our previous results in showed no double-sized product (~400 nt). Moreover, our results showed that the templates with oxidized 3′-OH exhibited the same ability for RNA synthesis initiation (, lanes 5 vs. 6), thereby excluding the possibility that FHV protein A uses the copy-back mechanism. Taken together, our results show that FHV protein A initiates RNA synthesis generally via a de novo mechanism.
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