IiPreventing Idle Ribosomes in the Presence of an Excess of EFG

We already mentioned that four proteins participate in translation during the elongation phase. Two ofthem, EF-Tu and EF-G, are components of the central activity of the ribosome, the elongation cycle, where in a cycle of reactions the nascent chain is extended by one amino acid (aa). EF-Tu brings the aa in the form of aa-tRNA to the decoding centre of the ribosome and EF-G translocates the tRNAs on the ribosome by one codon length (reviewed in ref. 20). These two factors, together with EF-Ts and EF4, belong to the superfamily of GTP-binding (G) proteins, which is an important class of regulatory proteins. They bind GDP or GTP and undergo a conformational change depending on the presence of which nucle-otide is bound. In the GTP conformer (the "on"-state) they bind to their target and promote a distinct reaction of the target, with the energy being paid by the binding energy generated during the binding step of the G protein. The target then signals that the reaction has been successfully accomplished and triggers the hydrolytic

Figure 2.6. Various ATP regeneration systems for in vitro protein expression. A) Conventional scheme for energy regeneration using phos-phoenol puryvate (PEP) and pyruvate kinase. The generated orthophosphate (Pi) inhibits protein synthesis. B) Improved energy regeneration using pyruvate, pyruvate oxidase and endogenous acetate kinase (according to ref. 19) C) Near in vivo system based on phosphoirans-acetylase (Pta) to produce acetylphosphate. The boxes in B and C show a common set of reactions for the regeneration of ATP. The PEP synthase (Pps) is blocked by the presence of oxalate. Ack, acetate kinase; Ldh, lactate dehydrogenase; Pdh, pyruvate dehydrogenase; Pyk, pyruvate kinase. (Modified from ref. 29).

Figure 2.6. Various ATP regeneration systems for in vitro protein expression. A) Conventional scheme for energy regeneration using phos-phoenol puryvate (PEP) and pyruvate kinase. The generated orthophosphate (Pi) inhibits protein synthesis. B) Improved energy regeneration using pyruvate, pyruvate oxidase and endogenous acetate kinase (according to ref. 19) C) Near in vivo system based on phosphoirans-acetylase (Pta) to produce acetylphosphate. The boxes in B and C show a common set of reactions for the regeneration of ATP. The PEP synthase (Pps) is blocked by the presence of oxalate. Ack, acetate kinase; Ldh, lactate dehydrogenase; Pdh, pyruvate dehydrogenase; Pyk, pyruvate kinase. (Modified from ref. 29).

reaction "GTP ^ GDP + Pi" in the corresponding enzymatic centre of the G protein. Upon release of the inorganic phosphate, the G protein switches into the GDP conformer ("off"-state) and, losing its affinity for the target, dissociates from it.

Usually a G protein recognizes a specific configureuration in the target before promoting or even triggering a specific reaction. This is also true for EF-G; it recognizes specifically the ribosome state before translocation and dissociates from the ribosome following GTP hydrolysis and Pi release. However, EF-G is the only G protein involved in translation that also interacts effectively with empty or idle ribosomes, probably because empty ribosomes seem to have "conformational memory" of the two main states before and after translocation.21 Thus empty ribosomes can trigger EF-G dependent GTP hydrolysis with a high turnover, which is not seen with EF-Tu. It follows that in a minimal system with poor energy resources, both ribosomes and EF-G need to be present in limiting amounts. This condition is attained when the molar ratio of EF-G:ribosomes is about 0.2 to 0.3, i.e., 2-3 EF-G molecules per 10 ribosomes.

2.7. Conclusion

In this chapter we have tried to estimate the minimal set of genes needed for a minimal living cell. A brave estimate of the minimal components for the translational apparatus today comprises no more than 200 genes, of which more than 120 are associated with the translational apparatus, encoding about 40 genes for ribosomal proteins, two rRNAs (omitting the 5S rRNA), 21 tRNAs, 20 synthetases, six factors and at least 20 tRNA modifying enzymes. In addition a minimum of30 genes are needed for both the generation of household energy and the synthesis of at least some of the amino acids (note: since some ofthe amino acids were formed in the Stanley Miller type experiments mimicking the atmosphere and the physical environment of more than 3 billion years ago,8 they could be taken up from the primordial soup by the earliest cells and thus did not need to be synthesized).

Even if we consider a "limping" life form, we end up with a total of at least 150 genes. Interestingly, this is in a reasonable agreement with the most restricted life form known today, namely the bacterium Carsonella ruddii with 182 genes.22 This bacterium is an obligatory endosymbiont living in phloem sap-feeding psyllids ("jumping plant lice"), in specialized cells (bacteriocytes) made by these insects. The species belongs to the y-proteobacteria as does Escherichia coli, is transmitted by vertical transmission through the host generations (no exogenous infections!), contains a full translation system and produces some amino acids for the host. We therefore think that it is not possible to construct a minimal cell "getting-by" with significantly less than 150 genes.

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