Continued protein synthesis at low [ATP] and [GTP] …

To measure the dependence of the protein synthesis rate on [ATP] and [GTP], we used an in vitro cell-free system derived from E. coli extracts. Cell-free systems provide the opportunity to directly monitor, add, and remove components with much greater ease and accuracy than can be done with in vivo systems. In addition, they have the potential to reproduce much of the complexity of in vivo metabolism without the threat that the catalytic system will be altered by continued synthesis of system components.

ATP-consuming process of protein synthesis

The energy for the synthesis of ATP comes from the breakdown of foods and phosphocreatine ..

Continued Protein Synthesis at Low [ATP] and [GTP] …

We used a prokaryotic combined transcription-translation system to show, for the first time, the relationship between protein synthesis rate and ATP concentration. We observed that the protein synthesis system exhibits much stronger ATP affinity than the ATP affinity suggested by characterization of individual enzymes. We also determined the dependence of protein synthesis rate on [GTP], which agrees with previous data for in vitro enzyme measurements and provides confidence in our data. The high affinity of the protein synthesis system for ATP and GTP underscores the ability of cells to remodel themselves upon a shift from high growth rates to energy-limited conditions. Moreover, this work demonstrates the importance of using cell-free biology as an integrated platform to obtain accurate information about the behavior of metabolic systems in which there are complex and often poorly understood interactions. We expect that this analytical approach will open the door for testing many hypotheses that deepen our understanding of the mechanisms controlling metabolic systems. For example, the same experimental system can be used to explore the effects of EC while a constant ATP concentration is maintained.

Novel Antibiotics Targeting Respiratory ATP Synthesis …

Since cell-free protein synthesis is emerging as a potential commercial recombinant DNA protein expression technology (, , ), it is also interesting that the modern systems do not appear to be energy limited. The measured ATP and GTP concentrations (200 μM or higher) are much higher than the Kms for protein synthesis (, , ).

Continued protein synthesis at low [ATP] and ..

While the low KmATP was unexpected if aminoacylation was rate limiting, another possibility is that while aminoacylation is the most energy-intensive step, it is not the limiting step. If elongation, for example, were the rate-limiting step under energy-limited conditions, then the rate of aminoacylation may be far lower than the maximum rate and still provide ample aminoacyl-tRNA for protein synthesis. Because tRNAs from a commercial source are added to the cell-free system, there are excess tRNAs relative to other translational components. This is consistent with the interpretation that surplus aminoacyl-tRNAs could accumulate if elongation was limiting. However, we observed that a lack of tRNA addition had a very minor effect on cell-free protein synthesis, decreasing yields by at most 5%. An interesting follow-up study might be to use a cell-free translation system reconstituted from purified elements (). Here, the levels of tRNAs could be fine-tuned without endogenous background levels. tRNA levels could be decreased, if necessary, until they were rate limiting, and the KmATP could be determined.

The uses of ATP? - The Student Room

Despite our attempt to mimic the cytoplasm, three aspects of the chemical environment remain nonphysiological. First, 2.4 mM Tris is carried over into the cell-free system as a result of extract preparation, adding a mild detergent. To date, our efforts to mimic the cytoplasm have focused on mimicking salts added directly to the cell-free protein synthesis system () without attempting to change the extract preparation procedure. Workers in our laboratory are now trying to develop new extract preparation protocols to target removal of Tris and to use glutamate salts rather than acetate salts in extract preparation. Second, 33 mM sodium is added to the reaction mixture as the cationic species complexed to pyruvate, introducing an extra parameter. We attempted to avoid the use of sodium pyruvate by using 33 mM pyruvic acid and adjusting the pH to 7.0 with potassium hydroxide or ammonium hydroxide. Unfortunately, the protein synthesis rates and yields decreased significantly under these conditions for unknown reasons. Recently, however, we discovered that the Cytomim system can be fueled by glutamate salts alone (). Using the pyruvate-free system could eliminate any possible effects of sodium on the system. The third nonphysiological aspect of the system is that phosphate concentrations are low. In the experimental apparatus used here, the lower and upper bounds of the phosphate concentration in the reaction mixture are between 0.5 and 5 mM (). Increasing the phosphate concentration may help activate phosphorylation reactions (e.g., nucleotide regeneration and metabolism) and phosphorolysis (). As shown in Fig. , nucleotide regeneration, phosphorolysis (where inorganic phosphate attacks mRNA), and mRNA degradation by RNases are important components of nucleotide metabolism that provide coupling between transcription, protein synthesis, and mRNA turnover (, ). Nucleotide metabolism is also relevant in central metabolism (e.g., in the regulation of glycolysis []) and in DNA synthesis, although these processes are not shown in Fig. . Adding higher concentrations of phosphate to the cell-free system may encourage more natural metabolism. For example, we recently showed that supplementing the pyruvate-free Cytomim system with 10 mM phosphate (the optimum experimental concentration) increased protein synthesis yields (). On the other hand, the low phosphate concentrations used here may have restricted ATP regeneration inside the reaction chamber and therefore may have been necessary to maintain low ATP concentrations. More closely emulating the cytoplasmic environment by addressing these three aspects of the chemical environment is expected to encourage more natural behavior and may impact our results. We believe that deviations caused by insufficient reproduction of in vivo conditions likely would have resulted in a KmATP higher than the true value. Thus, at a minimum, our data provide a conservative estimate of the KmATP for protein synthesis.

ATP (adenosine triphosphate) ..

The system-level KmATP, however, was unexpectedly low. Charging tRNAs with amino acids is the single most energy-demanding step in protein synthesis (), particularly when the fact that each mRNA is used multiple times is considered (). Therefore, we anticipated that the overall dependence of the rate on the ATP concentration might be controlled by the lowest ATP affinity reported for the aminoacyl-tRNA synthetases, which is on the order of 500 μM (). In contrast, the protein synthesis system exhibits much stronger ATP affinity (Table ). It is interesting that at an ATP concentration of 27 μM, such a synthetase would be expected to function at a rate that is only about 3% of the rate observed with the cytoplasmic ATP concentration present when the organism has an adequate energy source (3 mM). With 27 μM ATP the protein synthesis rate would still be 50% of the prestarvation rate.