Replicase Replication Complex

The RNA replication complex of a picornavims is bound to smooth cytoplasmic membranes (10, 16). In order to obtain a soluble RNA dependent activity it is necessary to dissociate the enzyme from the membranes by means which adversely affect the activity of the enzyme. It is also more laborious and costly to grow and infect large quantities of animal cells than to carry out a large scale infection of E. coli with an RNA bacteriophage. So far, because of these limitations, the isolation of a picornavims replicase was carried out with very dilute solutions of enzyme-protein, a... 

The work carried out so far on the EMC virus replicase provides a method for the isolation of minute quantities of an unstable RNA dependent replicase which allowed one to conduct a preliminary study of some of the properties of the enzyme. However, information on the ezyme s subunit composition, its mode of action, and on possible additional factors that may determine its specificity toward an EMC vims RNA template will require a stable enzyme preparation at a higher level of purity. There is also a growing feeling that the elucidation of the mechanism of biosynthesis of the picomaviral RNA will probably depend on an understanding of the possible functional relationships between the viral RNA replication complex and the smooth membranes in which it is enclosed. 

Initial translation of the genomic RNA produces the nonstruc-tural polypeptides for viral RNA replication at the rough endoplasmic reticulum (RER) membrane. RNA replication also occurs in association with the membranes of vacuoles which develop in the infected cells (Grimley et al., 1968). These cytopathic vacuoles appear to be formed by fusion of various preexisting vacuoles. The viral RNA replication complex probably contains nonstructural proteins which have replicase and transcriptase activities including RNA chain elon-... 

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... 

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. 

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-...

Other important questions remain to be solved are the single monomeric DNA polymerases detected in different species unique or do they correspond to a predominant activity in vitro which hides the activities of one or several other DNA polymerases (as DNA polymerase I hides DNA polymerases 11 and 111 in eubacteria) does this monomeric DNA polymerase correspond to a replicase (or to the catalytic subunit of a replicase complex), or to a repair enzyme The in vitro study of the replication of an extrachromosomal DNA would be useful for such investigations, but a definitive answer would require the isolation of conditionally lethal mutants. 

Fig. 15. RNA-phage infection of a bacterial cell as a simple hyper-cyclic process. Using the translation machinery (T) of the host cell the infectious plus strand (I ) first instructs the synthesis of a protein subunit which associates with three host proteins to form a phage-specific RNA-replicase. This replicase complex (R) exclusively recognizes both phage-RNA strands, plus and minus, and replicates them.

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