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An enzyme that catalyzes the conversion of a molecule into one of its isomers is an isomerase. This change from phosphoglucose to phosphofructose allows the eventual split of the sugar into two three-carbon molecules. Step 3. The third step is the phosphorylation of fructosephosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a high-energy phosphate to fructosephosphate, producing fructose-1,6-bisphosphate. In this pathway, phosphofructokinase is a rate-limiting enzyme.
This is a type of end-product inhibition, since ATP is the end product of glucose catabolism. Step 4.
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The newly-added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme, aldolase, to cleave 1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone-phosphate and glyceraldehydephosphate. Step 5. In the fifth step, an isomerase transforms the dihydroxyacetone-phosphate into its isomer, glyceraldehydephosphate. Thus, the pathway will continue with two molecules of a single isomer. At this point in the pathway, there is a net investment of energy from two ATP molecules in the breakdown of one glucose molecule. One possible mechanism for adaptation to stressful conditions is genetic change that produces novel traits or new physiological functions, such as antibiotic resistance or the ability to use new metabolic pathways Blount et al.
Another mechanism is genetic change that confers restoration of cellular functions to a prestressed state. This pattern of restoration has been recently observed during metabolic perturbation and high-temperature adaptation Fong et al. Although both novelty and restoration likely drive long-term adaptation, it is not yet clear which is more frequent. Another issue that remains largely unexplored is the temporal pace of phenotypic change during adaptation to stress.
After an environment becomes stressful, the acclimated state becomes the initial phenotypic state upon which natural selection acts.
Thereafter, each adaptive mutation moves the population toward a phenotypic optimum i. For historical and methodological reasons Orr , most evolutionary studies have focused on the end product of adaptation, leaving the early and intermediate steps of adaptation largely unexplored. Fortunately, studies on experimental evolution may provide insight into the sequential magnitude of fitness changes during adaptation.
In contrast, subsequent mutations confer more moderate fitness gains Chou et al. Although our knowledge about the temporal trajectory of fitness continues to grow, few studies have examined shifts in phenotypes during the adaptive process. One exception is the study of Fong et al. Fong et al.
Most of the GE changes during adaptation were restorative Fong et al. Unfortunately, however, the genetic bases of these phenotypic changes were not resolved; it was unclear if the changes in GE during the intermediate point of adaptation were caused by an early adaptive mutation or were caused by combinations of mutations that accumulated during the experiment. Thus, many questions regarding phenotypic adaptation remain unresolved, such as the following: What are the molecular effects underlying the large fitness advantage conferred by first-step mutations? And, what is the phenotypic contribution of the first-step mutation compared with later steps in an adaptive walk?
We have decided to explore these questions based on our recent, large-scale evolution experiment Tenaillon et al. After 2, generations, we isolated single clones from each evolved population and identified the genetic changes that had accumulated during the experiment. Furthermore, these mutations typically appeared early in the evolution experiment.
Yet, some questions remain. First, we have not identified the mechanistic bases of their fitness advantage.
Steps of Genetic Transcription | Biology for Majors I
That is, it is still unclear if these mutations confer the same phenotypic effects as rpoB mutations found in other regions of RNAP that were also selected in some lineages during the thermal adaptation experiment. In this study, we address all these questions. Solving these questions is relevant for understanding the adaptive mechanisms prevalent in our experimental conditions and also in other evolutionary experiments, because mutations that affect transcriptional regulators such as RNAP and the Rho termination factor typically appear in the early stages of stress adaptation Applebee et al.
Numerous observations point to the possibility that highly pleiotropic mutations in transcriptional regulators could be the first step of a general mechanism of adaptation Fong et al. With these considerations in mind, we have investigated the timeline of adaptive change during our evolution experiment fig. We then explore first-step mutations in terms of their effect on growth and GE relative to the ancestral strain.
More specifically, we explore whether three amino acid substitutions in the RNAP active site converge on the same expression phenotype and also whether their effects are similar to another rpoB mutation that affects an amino acid substitution far from the RNAP active site.
Finally, we evaluated the phenotypic contribution of one of these first-step mutations relative to the end product of adaptation, as represented by two clones from the end of the 2, generation experiment.
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Thermal stress adaptation timeline. The null hypothesis is that the mean of the ancestor and the mean of the mutant are equal. Of the genes involved in translation, 54 genes transcribed products that were structural constituents of ribosomes GO: , including rpl , rpm, and rps genes supplementary table S1 , Supplementary Material online. Surprisingly, the heat shock inducible genes Nonaka et al.
Of the 66 genes that had been identified previously as a component of the general stress response Weber et al. To be sure that this GE profile did not result from sampling at the wrong phase of the growth curve, we repeated the experiment. We again sampled three replicates at midexponential, repeated RNA sampling, and transcriptome analyses.
We complemented this observation by estimating two components of fitness—the duration of the lag phase and the maximum growth rate. We also calculated the final yield, which is not a component of fitness Vasi et al. We note, however, that an alternative definition of lag phase, which was interpolated from the maximum growth rate see Materials and Methods , reflects a shorter but not significantly different lag phase in the mutants supplementary table S3 , Supplementary Material online.
Our second, nonexclusive hypothesis was that the mutated RNAP led directly or indirectly to altered transcription of a set of genes and this differential expression underlies growth improvement Conrad et al.
In turn, we reasoned that the reduced speed of RNAP increases both transcription fidelity and termination efficiency Jin et al. In our experiment, transcription efficiency captures both the speed and accuracy of all events during transcription, including initiation, elongation, and termination Reynolds To measure the transcription efficiency we measured the relative abundance of an inducible fluorescent gene YFP inserted in our strains, at different points post induction see Materials and Methods.
Using this method Reynolds ; Brandis et al. S1 , Supplementary Material online. Finally, the mutant IN had 1, DEGs with highly downregulated and highly upregulated genes. Within the confines of this experiment, we cannot differentiate as to whether these changes in GE were caused by direct effects, such as modified binding of the RNAP, or indirect, downstream effects.
Nonetheless, it is clear that single mutations within codon of rpo B lead to numerous, pleiotropic shifts in GE. To investigate the general trend in GE changes from the ancestor to acclimation and then from acclimation to first-step mutations fig. The first was Principal Component Analysis, which simultaneously considered the expression of all genes and all clones.
S2 , Supplementary Material online. Global changes in GE during the acclimation and adaptive response. Changes in expression were categorized and colored as follows: Restored blue , reinforced red , and novel yellow. Both unrestored and uninformative genes are colored in grey. The black line represents the linear regression fitted to the dots in each graph. We next examined the GE changes in more detail by characterizing the expression of individual genes into one of the four patterns of change that denote the direction of the effect of the mutated RNAP see Materials and Methods and supplementary table S4 , Supplementary Material online.
For these definitions, we investigated genes that differed in expression either during acclimation or between acclimation and first-mutation clones supplementary table S4 , Supplementary Material online ; as a result, we categorized directional shifts of roughly half of 4, E. Finally, we based our analyses on two replicates of each of our single mutant clones. However, the broad similarity in results across the 3 codon mutations suggest that the 3 clones can be treated as pseudoreplicates.
Accordingly, we combined the six replicates two from each of the three single mutations and reran GE analyses to examine the effect of increased sampling.
Thus, although the statistical power to detect differences likely varies among clones, the combined data reinforce the observation that a large proportion of genes were restored in GE by codon rpoB mutants. Given that rpoB mutations in codon converged in phenotype toward restorative GE, we sought to know if additional mutations in rpoB had similar effects.
The IS mutation was found in 15 of our high-temperature adapted clones Tenaillon et al. That is, the IS mutation tended to restore GE from the acclimated state toward the prestressed state fig. This high level of phenotypic convergence was also highlighted by the pairwise comparisons of differential expression between the mutants supplementary fig. S3 , Supplementary Material online. We performed an enrichment analysis of GO assignments for the restored genes that were shared among the 4 mutants.