Browsing by Author "Arnold, Sabine"

Filter results by typing the first few letters
Now showing 1 - 1 of 1
  • Thumbnail Image
    ItemOpen Access
    Kinetic modelling of gene expression : from linear genome sequence to nonlinear cellular dynamics
    (2003) Arnold, Sabine; Reuss, Matthias (Prof. Dr.-Ing.)
    In this study, a dynamic model of prokaryotic gene expression was developed that heavily makes use of gene sequence information. The main contribution arises from the fact that the combined gene expression model allows to assess mechanistically the impact of nucleotide sequence alteration on the dynamics of gene expression rates. Modelling required the development of a valid model structure for template-bound biopolymerization processes within a continuous analysis method. In contrast to a discrete model, or a combination of both approaches (i.e., hybrid modelling), the continuous model presented is a mechanism-based deterministic description of system states in terms of differential and algebraic sets of equations. Characteristically, a codon-specific representation of state variables was chosen for this model. Transcription kinetics were described mathematically at the example of T7 RNA polymerase. Parametrization of the transcription model was carried out for selected model constants, i.e., for the rate constants of initiation, elongation, and termination, as well as for the maximum rate of transcription. According to enzyme kinetics, most influential parameters determining transcription rate are T7 RNA polymerase concentration, and promoter concentration at typical reaction conditions of simultaneous in vitro transcription/translation. The process of mRNA degradation was modelled allowing for a distinction between endonucleolytic and exonucleolytic reaction steps. The effects of increased translational efficiency greatly improving mRNA stability, as observed experimentally, were correctly demonstrated by the model. On the basis of simulating lacZ mRNA degradation, it was possible to identify the parameters contained in the degradation model. The translation model presented covers the mechanisms of protein synthesis initiation, elongation, and termination, at the same time considering the particular mechanistic role of key translation factors. An earlier approach for describing sterical interference among template-bound catalysts (MacDonald et al., 1968) was extended in this study, in order to cover also a situation where two different types of catalysts (i.e., ribosomes and degradosome) can be bound in multiple copies to a same template. The number of state variables could be significantly diminished by merging groups of base tripletts together (model reduction), while at the same time taking into account the implications on reaction kinetics and material balancing. The current status of the combined model allows to reveal several causes for production limitation, whether they are due to substrate depletion or inactivation processes, or else caused by unfavourable initial catalyst concentrations and their stoichiometric relations. An application of the combined gene expression model to simulating cell-free protein synthesis dynamics demonstrated limited volumetric productivitites to be caused by unfavourably low translation factor levels that are typical for these dilute in vitro systems. Equilibrium binding calculations suggested a requirement for at minimum equal molar ratios of initation factors IF1, IF2, and IF3, with respect to total concentration of unbound ribosomes. By appropriately raising the concentrations of both translation initiation and elongation factors, a 4-fold improvement of volumetric protein synthesis rate and a 5-fold higher final product yield are predicted in comparison to a non-optimized reference batch process. From a stand-point of reduced model complexity, it may be beneficial to use the overall model for estimation of mechanism-related parameters or decay constants of a gene expression model, prior to applying these parameters within a whole-systems modelling frame. The immediate value of such models arises from their applicability for describing the expression of individual genes or a few genes at a time, which are typical for recombinant protein production. Gene sequence information enters the overall model at the following stages: (a) within the transcription process, by assigning different rate constants for initiation and termination of mRNA synthesis, respectively, (b) the endo- and exonuclease activities in the ordered process of 5' to 3'-degradation of messenger RNA, (c) during translation by distinguishing codon-specific elongation rates and effects related to sterical interactions among translating ribosomes. An attempt was made also to investigate the impact of predicted mRNA secondary structure within the ribosome binding site on measured translation rate. The results of this analysis suggest improved protein synthesis rates for mRNA conformations that favour a high degree of single-strandedness within a region of approximately 20 nucleotides downstream of the Shine-Dalgarno sequence. Coincidently, this mRNA region is covered by the ribosome during the event of ribosome binding.