RNA replicase

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). 

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. 10.9 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)... 

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.)...

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. 

The RNA replicase of most RNA bacteriophages has a molecular weight of -210,000 and consists of four subunits. 

These three host proteins may help the RNA replicase locate and bind to the 3 ends of the viral RNAs. 

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. 

As we shall see in the next chapter, some natural RNA molecules catalyze the formation of peptide bonds, offering an idea of how the RNA world might have been transformed by the greater catalytic potential of proteins. The synthesis of proteins would have been a major event in the evolution of the RNA world, but would also have hastened its demise. The informationcarrying role of RNA may have passed to DNA because DNA is chemically more stable. RNA replicase and reverse transcriptase may be modem versions of enzymes that once played important roles in making the transition to the modern DNA-based system. 

Coding versus Template Strands The RNA genome of phage Q/3 is the nontemplate or coding strand, and when introduced into the cell it functions as an mRNA. Suppose the RNA replicase of phage Q/3 synthesized primarily template-strand RNA and uniquely incorporated this, rather than nontemplate strands, into the viral particles. What would be the fate of the template strands when they entered a new cell What enzyme would such a template-strand virus need to include in the viral particles for successful invasion of a host cell ... 

The Chemistry of Nucleic Acid Biosynthesis Describe three properties common to the reactions catalyzed by DNA polymerase, RNA polymerase, reverse transcriptase, and RNA replicase. How is the enzyme polynucleotide phos-phorylase similar to and different from these three enzymes ... 

RNA catalysis is not only concerned with RNA cleavage non-natural ribozymes that show ligase activity (Bartel and Szostak, 1993) were obtained and many (so far not yet successful) efforts have been undertaken to prepare a ribozyme with RNA replicase activity. RNA catalysis does not only operate on RNA, nor do nucleic acid catalysts require the ribose backbone. Ribozymes were trained by evolutionary techniques to process DNA rather than their natural RNA substrate (Beaudry and Joyce, 1992), and catalytically active DNA molecules were evolved as well (Breaker and Joyce, 1994 Cuenoud and Szostak, 1995). Systematic studies revealed many other examples of RNA catalysis on non-nucleic acid substrates (see... 

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... 

Let us come therefore to the basic concept of the paradigm, i.e. to the idea that the smallest replicative system is simpler that the smallest metabolic system. This is the problem that we need to address, and in order to do so we must first answer a preliminary question what is the smallest system that allows the replication of RNAs The answer has come from two classic experiments, one by Sol Spiegelman in 1967 and the other by Manfred Eigen in 1971. In both cases the environmental conditions were simplified to the highest degree, and the experiments were performed in solutions containing free nucleotides and RNA-replicase enzymes. 

So far, we have constructed an unsatisfying picture of the earliest days of an RNA world although some prebiotic mechanisms may exist for the untemplated formation of oligonucleotides, these molecules would have been short, would have contained a variety of monomers besides ribotides, and could not have been faithfully copied by the template-directed polymerization of monomers. Given this model, it is difficult to imagine the accumulation of RNA sequences necessary for the Darwinian selection of a multitude of active ribozymes. Nevertheless, these precursors may have been adequate for the first critical step in the formation of life the formation of an RNA replicase. 

Santoro and Joyce (218). The last authors cited reported the selection of multipurpose RNA-cleaving DNAzymes with potential applications in molecular biology. The elusive RNA replicases, which should be able to replicate RNA structures including their own to sustain the RNA world hypothesis, are a common target for research a recent review (219) summarized the efforts in this field. 

Figure 26. In serial dilution experiment, Spiegelman [73, 74] and co-workers obtained three successive mutants with increased adaptation to presence of ethidium bromide, a drug that interferes with replication. Experiment starts with population of 10 MDV strands (variant of Q -RNA comprising about 220 nucleotides that is well-adapted to Q -RNA-replicase). Population is amplified to about 10 copies and subsequently diluted to initial concentration. Iteration of this procedure led to final product, three-error mutant that was obtained after about 40 iterations. As replication rate data show, mutant is slightly inferior to wild type in absence of ethidium bromide but twice as efficient as wild type at final concentration of ethidium bromide.

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

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