Replicase replicator

RNA Viruses The Replication of RNA Genomes RNA-Dependent RNA Replicases Replication of Retroviral Genomes (Figure 24.45)... 

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)... 

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. 

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.)...

FIGURE 27. General kinetic mechanism of DNA replication. E is the replicase (polymerase), D the growing DNA replicant, N is MgdNTP and P represents MgPPi. Adapted with permission from Reference 290. Copyright 2006 American Chemical Society... 

Replication in E. coli requires not just a single DNA polymerase but 20 or more different enzymes and proteins, each performing a specific task. The entire complex has been termed the DNA replicase system or replisome. The enzymatic complexity of replication reflects the constraints imposed by the structure of DNA... 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

One subunit (Mr 65,000) is the product of the replicase gene encoded by the viral RNA and has the active site for replication. The other three subunits are host proteins normally involved in host-cell protein synthesis the E. coli elongation factors Tu (Mt 30,000) and Ts (Mr 45,000)... 

RNA replicase isolated from Qj8-infected E. coli cells catalyzes the formation of an RNA complementary to the viral RNA, in a reaction equivalent to that catalyzed by DNA-dependent RNA polymerases. New RNA strand synthesis proceeds in the 5 —>3 direction by a chemical mechanism identical to that used in all other nucleic acid synthetic reactions that require a template. RNA replicase requires RNA as its template and will not function with DNA. It lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase. Unlike the DNA and RNA polymerases, RNA replicases are specific for the RNA of their own virus the RNAs of the host cell are generally not replicated. This explains how RNA viruses are preferentially replicated in the host cell, which contains many other types of RNA. 

Fig. 2- A characteristic of the mechanism of RN A replication is the coupled pair of cycles of synthesis, for the plus- and minus-strand, respectively. A catalytically active complex consists of the enzyme (replicase) and an RNA template. Four phases of each cycle can be distinguished (I) the commencement of replication by the binding of at least two substrate (nucleoside triphosphate) molecules (2) elongation of the replica strand by successive incorporation of nucleotides (3) dissociation of the complete replica away from the replicase (4) dissociation of the enzyme from the S end of the template and its reassociation with the 3 end of a new template. The matrix is represented by I (information), the enzyme by E, and the reaction product by P. The ultimate reaction product P is then used as a template (I). The substrate, S, is the triphosphate of one of the four nucleosides A, U, G, and C. 

Fig. 4. A solution of nucleotide triphosphate is incubated in the presence of QP replicase for just long enough to assure the manifold replication of any templates that may contaminate the enzyme. The incubation is interrupted before even one template has time to arise de novo. The solution is then divided up into portions and the incubation is continued, this time long enough to allow products to arise de novo and to multiply. The RNA formed in each portion is analyzed by the fingerprint method various different reaction products are found. Sometimes the growth curve displays the appearance of a new mutant. Although the incubation time of template-instructed synthesis is determined unambiguously (because of the superposition of many individual processes) the synthesis de novo shows a scatter of induction times. This indicates that the initiation step is a unique molecular process which is then rapidly amplified. ...

The first successful attempts to study RNA evolution in vitro were carried out in the late sixties by Sol Spiegelman9 and his group at Columbia University (Spiegel-man, 1971). They made use of an RNA replicase isolated from Escherichia coli cells infected by the RNA bacteriophage QP and prepared a medium for replication by adding the four ribonucleoside triphosphates (GTP, ATP, CTP, and UTP) in a suitable buffer solution. QP RNA, when transferred into this medium, instantaneously started to replicate. Evolutionary experiments were carried out by means of the serial transfer technique (Figure 4). Materials consumed in RNA replication... 

Figure 4. The technique of serial transfer. An RNA sample which is capable of replication in the assay is transferred into a test-tube containing stock solution. This medium contains the four nucleoside triphosphates (ATP, UTP, GTP and CTPJand a virus specific RNA polymerase, commonly QP-replicase because of the stability of this protein, in a suitable buffer solution. RNA replication starts instantaneously. After a given period of time a small sample is transferred to the next test-tube and this procedure is repeated about one hundred times. The transfer has two consequences (i) the material consumed in the replication is replaced, and (ii) the distribution of RNA variants is subjected to a constraint selecting for the fastest replicating species. Indeed, the rate of replication is increased by several orders of magnitude in serial transfer experiments starting out from natural QB RNA and leading to variants that are exclusively suited for fast replication and hence are unable to infect their natural hosts, Escherichia coli.

Figure 5. The mechanism of RNA replication by means of viral-specific RNA repli-cases. The sketch shows a simplified version of the mechanism of RNA replication by QP replicase. The mechanism within each of the two cycles consists of binding of the RNA (l+ and I" standing for plus- or minus-strand respectively) to the enzyme (E), elongation of the growing chain (see Figure 2) and, eventually, dissociation of the enzyme-RNA complexes. Rate constants for association, elongation and dissociation are indicated by kX, kf, kp, k), ki and kp, respectively. According to complementar-...

Beaudry, A.A. Joyce, G.F. (1992). Directed evolution of an RNA enzyme. Science 257,635-641. Biebricher, C.K. (1987). Replication and evolution of short-chained RNA species by Q 3 replicase. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 52, pp. 299-306. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 

In a similar vain, Szostak et al. [17] propose that a protocell composed of a growing membrane, a general replicase ribozyme (able to replicate also another copy of itself), and another ribozyme involved at some stage in membrane formation would be truly alive. Once again, it is clear that this system is an ultimate heterotroph [19], completely devoid of a metabolic sub-... 

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