Replicase membranes

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 ). 

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.)...

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-... 

Spiegelman s RNA system vHth a replicase enzyme is very artificial there is only one molecule being reproduced. A living cell has many constituents, and part of the competition in evolution involves which cell has the best mixture of constituents. The whole organism must evolve, with all its parts. This cannot happen in a soup it requires individuals surrounded by a barrier, hence a membrane. 

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... 

Caliguiri and Tamm (I6) have shown that in HeLa cells infected with poliovirus the replicase is associated with the smooth cytoplasmic membranes. Since a similar situation is found in the case of the EMC replicase in infected BHK cells, in the next step the smooth cytoplasmic membranes are isolated from the 20,000g supernatant by fractionation in a 20 to 60% discontinuous sucrose gradient. The distribution of the replicase activity in the sucrose gradient layers is shown in Table 1. The greater part of the activity is found in the second layer from the top (fraction 2) at a sucrose concentration of 18%. (When uninfected cells are homogenized and fractionated in the same way, fraction 2 is foiind to contain the smooth membranes of the endoplasmic reticulum). Fraction 2 contains only a small amount of the total protein, 30% of the phospholipids and about 50-80% of the replicase activity. 

Infection of HeLa cells with poliovirus elicits extensive proliferation of smooth cytoplasmic membranes (16). This occurs also in BHK cells infected with EMC virus. The results of an experiment in which smooth cytoplasmic membranes were isolated from aliquots of infected cells at various times after infection are shown in Figure 2. The membrane pellets were assayed for activity of EMC replicase and the protein and phospholipid content determined. For the first 4 hours of infection there is no change in the amount of celliilar smooth membranes. However, between 4 and 8 hours, at the time when the viral replicase is being formed, there occurs also a steep rise in the formation of smooth membranes. The protein content of the smooth membranes rises by about five-fold and the phospholipids by ten-fold. The diagram in Figure 5 depicts the increase of membrane material during infection and indicates also the changing ratios of protein to phospholipid which takes place in the smooth membranes synthesized between 4 and 8 hours after infection from 5.0 to 1.7. The low protein content of the new... 

Figarre 2. Time course of formation of smooth cytoplasmic membranes and EMC replicase in BHK cells infected with EMC virus. Smooth membranes were isolated from aliquots of 1.5 x 1o9 cells and analyzed for protein, phospholipid and replicase activity. 

At 4 hf 2 mCi of (55s) methionine were added, and the incubation continued to 6.5 h. The cells were collected and resuspended in a medixmi containing cold methionine. After a 1 h chase the cells were harvested and from them we prepared a whole cell lysate, isolated the smooth cytoplasmic membranes and from these purified the replicase by QAE Sephadex chromatography and glycerol gradient sedimentation. Samples of radioactive cell lysate, smooth membrane and purified enzyme were analyzed by SBS polyacrylamide gel electrophoresis. A profile of the radioactive proteins in a cell lysate is shown in Figure 10A. It contains three minor peaks of molecular weight 95>000> 75>000 and 65,000 which correspond to EMC virus unstable proteins B,B and Bl three of the four coat proteins a (34,000), 3 (30,000) and y (26,000) and relatively large amounts of stable EMC virus proteins E (56,000) and F (38,000). A profile of the EMC proteins found in the isolated smooth membranes is shown... 

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. 

Finally, mixing experiments were performed to examine whether anything inhibitory exists in the MDBK extract or stimulatory in the L-cell extracts. These experiments were done with both membrane-bound and soluble replicase. Results from these experiments indicated that there was no inhibitory factor in the MDBK extracts, nor stimulatory factors in the L-cell extracts. 

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-... 

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