Replicase

Figure C2.14.1. Diagram of a fragment of a folded RNA polymer, the QP replicase MDV-1 [176]. Note the various stmctural features stems closed with a loop ( hairjDins ), bows, and single strands.

In analogy with the designation of NRTIs and NNRTls for the nucleoside and nonnucleoside type of reverse transcriptase (RT) inhibitors to target HIV, the corresponding inhibitors to target HCV may be termed NRRIs (for nucleoside RNA replicase inhibitors) and NNRRIs (for nonnucleoside RNA replicase inhibitors). 

How do these NRRIs interact with their final target, the HCV RNA replicase They are phosphorylated to their 5 -triphosphate form, and then inhibit the HCV replicase. As they possess a 3 -hydroxyl function, they may not be considered as obligate chain terminators, but they may act as virtual chain terminators, viz. by steric hindrance exerted by the neighboring 2 -C-methyl and/or 4 -C-azido groups. Similar to their NRTI and NNRTI counterparts in the case of HIV reverse transcriptase, the NRRIs (2 -C-methylnucleosides) interact, upon their phosphorylation to the corresponding 5 -triphosphates, with a region of the HCV RNA replicase (or NS5B RNA-dependent RNA polymerase) that is clearly distinct from the site(s) of interaction of the NNRRIs (Tomei et al. 2005). 

We note in passing that DNA-refilicase has an essential thiol group which is labelled by mercurials. To our knowledge no studies of the effect of platinum on the replicase have been made and this is clearly an omission which should be rectified. In some proteins platinum derivatives would seem to go for the same sites as mercury(II) reagents — a not surprising result as their chemistry is similar. 

As noted, the viral RNA is of the plus (+) sense. Replicase synthesizes RNA of minus (-) sense using the infecting RNA as template. After minus RNA has been synthesized, plus RNA is made from this minus RNA. The newly made plus RNA strands now serve as messengers for virus protein synthesis. The gene for the maturation protein is at the 5 end of the RNA. Translation of the gene coding for the maturation protein (needed in only one copy per virus particle), occurs only from the newly formed plus-strand RNA as... 

Zeng, C.Q., Wentz, M.J., Cohen, J. et al. (1996) Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. Journal of Virology, 70 (5), 2736-2742. 

Fig. 8.4 Hypercycle phenomena can be observed when a cell is infected by an RNA virus. The vims provides the host cell with information for an enzyme favouring only the reproduction of viral information, i.e., of an RNA strand. This RNA is converted by the host cell into a protein (a replicase) which forms a new RNA minus-strand. The latter is then replicated to give a plus-strand (Eigen et al., 1982)...

Szostak et al. worked on the basis of a simple cellular system which can replicate itself autonomously and which is subject to Darwinian evolution. This simple protocell consists of an RNA replicase, which replicates in a self-replicating vesicle. If this system can take up small molecules from its environment (a type of feeding ), i.e., precursors which are required for membrane construction and RNA synthesis, the protocells will grow and divide. The result should be the formation of improved replicases. Improved chances of survival are only likely if a sequence, coded by RNA, leads to better growth or replication of membrane components, e.g., by means of a ribozyme which catalyses the synthesis of amphiphilic lipids (Figs. 10.8 and 10.9). We can expect further important advances in the near future from this combination ( RNA + lipid world ). 

Fig. 10.8 The importance of the vesicle for the Darwinian evolution of a replicase. Compart-mentalisation ensures that related molecules tend to stay together. This permits superior mutant replicases (grey) to replicate more effectively than the parent (black) replicases. The evolutionary advantage spreads in the form of vesicles with superior replicase molecules, leading with a greater probability to vesicles with at least two replicase molecules (or a replicase and a matrix molecule). Vesicles with less than two replicase molecules are struck out their progeny cannot continue the RNA self-replication. Thus, the vesicles with better replicases form the growing fraction of vesicles which carry forward the replicase activity (Szostak et al., 2001)...

Fig. 10.9 Possible reaction pathway for the formation of a cell. The important precursors are an RNA replicase and a self-replicating vesicle. The combination of these two in a protocell leads to a rapid, evolutionary optimisation of the replicase. The cellular structure is completed if an RNA-coded molecular species, for example, a lipid-synthesised ribozyme, is added to the system (Szostak et al., 2001)... 

An amplification system that actually amplifies exponentially RNA probe sequences bound to the target sequence, in contrast to PCR and TAS systems, which amplify target sequences, is the Q-beta replicase system (B4). Although this system can achieve a million- to billionfold amplification in 15 minutes at 37°C, background signal due to nonhybridized probes is reported to be very high. 

Shah, J. S., Liu, J., Buxton, D., Hendricks, A., Robinson, L. et al., Q-beta replicase-amplified assay for detection of Mycobacterium tuberculosis directly from clinical specimens. J. Clin. Microbiol. 33, 1435-1441 (1995). 

Sequencing.—With the elucidation of the primary and secondary structure of the replicase gene, the complete 3569-nucleotide-long sequence of the RNA of bacteriophage MS2 is now known.153 This is the first organism for which theentire nucleic acid structure has been elucidated, and Fiers and his group richly deserve their bouquet. 

DNA-directed RNA polymerase [EC 2.1.1.6] catalyzes the DNA-template-directed extension of the 3 -end of an RNA strand by one nucleotide at a time thus, n nucleoside triphosphate generate RNA and n pyrophosphate. The enzyme can initiate a chain de novo. Three forms of the enzyme have been distinguished in eukaryotes on the basis of sensitivity of a-amanitin and the type of RNA synthesized. See also Replicase... 

MICROTUBULE ASSEMBLY KINETICS RITCHIE EQUATION RNA, exonucleolytic cleavage, PHOSPHODIESTERASES RNA LIGASE RNA POLYMERASES EDITING MECHANISMS REPLICASE RNA stability,... 

Sutton, G., et al. (2004). The nsp9 replicase protein of SARS-coronavirus, structure and functional insights. Structure (Camb) 12,341-353. 

Fig. 8. The replicase reaction cycle with a focus on the native chemical ligation.

Fig. 9. (a) Melting points and (b) replicase turnover of the Leu9-modified peptides. Parental peptide (filled hexagons) other peptides with the following substitutions EGly (O), DfeGly (a), TfeGly ( ). 

The a-helical coiled coil-based screening system already provided a wide variety of information about the interactions of fluorinated amino acids within hydrophobic and hydrophilic protein environments. Investigations on the thermal stability as well as the replicase activity have both emphasized the orthogonal properties of fluorinated aliphatic amino acid side chains. The term orthogonal in this context has been chosen by us to demonstrate that they are in fact hydrophobic... 

In this regard, let us draw some lessons from contemporary RNA replication RNA self-replicase does not exist in nature, the actual concentrations of (2P repli-case and template-RNA in a single cell may be considered, and compare with in vitro experiments (Szathm and Luisi, unpublished data). Based on the smallest dimension of a bacterium, a minimal concentration of c. 10 nM can be calculated for RNA in vivo. 

Figure 7.11 Principle of a minimal replicase. (Adapted from Maynard-Smith and Szathmary, 1995. See also, with comments, Burmeister, 1998.)...

This is illustrated in Figure 11.3. It consists of a vesicle containing two ribozymes, one (Rib-2) capable of catalyzing the synthesis of the membrane component the other (Ribl) being an RNA replicase that is capable of repUcating itself, and reproducing the Rib-2 as well. In this way, there is a concerted shell-and-core replication, and there is therefore a basic metabolism, self-reproduction, and - since the replication mechanism is based on RNA replication - also evolvability. 

Figure 11.4 The hypothetical pathway for the transformation of a simple RNA cell into a minimal DNA/protein cell. At the first step, the cell contains two ribozymes, Rib-1 and Rib-2 Rib-1 is a RNA replicase capable of reproducing itself and making copies of Rib-2, a ribozyme capable of synthesizing the cell membrane by converting precursor A to surfactant S. During replication, Rib-1 is capable of evolving into novel ribozymes that make the peptide bond (Rib-3) or DNA (Rib-4). In this illustration, these two mutations are assumed to take place in different compartments, which then fuse with each other to yield a protein/DNA minimal cell. Of course, a scheme can be proposed in which both Rib-3 and Rib-4 are generated in the same compartment. (Modified fromLuisi et al., 2002.)...

Figure 11.8 Replication of RNA in self-reproducing vesicles. The initial vesicles contained the enzyme Q(3 replicase and the four ribonucleotides in excess, as well as the RNA template (the MDV-1 template). The division of vesicles is induced by the addition of oleic acid anhydride and the duplication of the figure is idealized, as in reality division occurs on a statistical basis. (Adapted from Oberholzer etal, 1995b.)...

An overview of the work in this field is presented in Table 11.5, see also recent reviews (Luisi et al, 2006). This table also contains references to the work mentioned earlier, such as poly(A) synthesis from ADP the PCR reaction in liposomes the RNA synthesis by QP replicase, as well as the expression of poly(Phe) by an entrapped ribosomal system. This work is preliminary to protein expression in liposomes. Going from here to the protein synthesis, it may be useful to compare the different strategies for the expression of GFP. 

Oleic acid/oleate vesicles containing the enzyme Q(3 replicase, the RNA template and the ribonucleotides. The water-insoluble oleic anhydride was added externally. 

Biebricher, K., Eigen, M., and Luce, R. (1981). Kinetic analysis of template, instructed and de novo RNA synthesis by Qbeta replicase. J. Mol. Biol, 148, 391 10. 

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