Figure 5.

Model 1 with autoregulation of MatK reproduces the observed expression dynamics. Two possible models of matK gene expression were analyzed with respect to how well both models fit to our experimental data. A, Reaction scheme of the tRNA-MatK splicing network for model 1. The MatK transtargets trnA, trnV, and trnI are lumped into one tRNA species assigned as tRNA_sum. The values of trnA accumulation (Fig. 3A) were used to describe the expression dynamics of tRNA_sum, and tRNA_sum is assumed to be 10-fold more abundant than trnK (for details, see “Results”). k_1r and k_5r are the transcription rates of the tRNA precursors pre-trnK-matK (y1) and pre-tRNA_sum (y5), respectively. The protein MatK (y4) is encoded within the intron of the trnK-matK precursor gene and translated with the rate k_4p. As the levels of the tRNA precursors y1 and y5 as well as the MatK protein y4 increase, they form pre-trnK/MatK (y2) and pre-tRNA_sum/MatK complexes (y6), respectively. The pre-trnK/MatK repression complex (y2) inhibits the translation of matK via a negative feedback loop (red line with a blunt end). The subscript τ in k_2aτ and k_6aτ is the delay introduced in the models to signify the time taken for the formation of pre-trnK/MatK and pre-tRNA_sum/MatK complexes at day 25 of tobacco development. Within the tRNA/protein complexes, MatK splices the introns with rates k_3s and k_7s, leading to spliced tRNAs: mature trnK (y3) and mature tRNA_sum (y7). Competition of pre-trnK-matK (y1) and tRNA_sum (y5) for MatK (y4) is assigned by the factors and . To simplify the model, the involvement of nucleus-encoded splicing factors was not considered. Complex dissociation was assumed to be slow and, therefore, was neglected. Dashed arrows represent the degradation of RNAs, complexes, and the MatK protein with rates k_1d to k_7d. B, Accumulation of tRNA precursors, mature tRNAs, MatK protein, and tRNA/MatK complexes during tobacco development, derived from theoretical predictions of model 1 in comparison with northern-blot and western-blot analyses (E). Model 1 (top) fits certain experimental data displayed in E, such as the inverse correlation of MatK expression and matK mRNA (pre-matK-trnK) accumulation around day 7, the sharp decline of the MatK protein level after day 7, and the enrichment of pre-trnK/MatK complexes in mature seedlings. The model consistently reproduces the ratio-to-ratio value between pre-trnK/MatK complexes at days 25 and 7 (RR_25d/7d = 1.24), indicated with black arrows. Model 1 (bottom) shows temporal expression profiles of tRNA_sum (trnA, trnV, and trnI). The days of maximal accumulation of tRNA_sum are the same as those for trnK and those experimentally observed. C, Model 2 shows the same reaction scheme depicted in A, but negative autoregulation is removed from the system. D, Accumulation of tRNA precursors, mature tRNAs, MatK protein, and tRNA/MatK complexes during tobacco development derived from theoretical predictions of model 2 (C) in comparison with northern-blot and western-blot analyses (E). Model 2 (without autoregulation) fails to capture the distinctive expression dynamics of matK mRNA (pre-trnK-matK) and MatK protein. E, Experimental data for the accumulation of tRNA precursors, mature tRNAs, and MatK protein during tobacco development derived from northern-blot and western-blot analyses. The accumulation of MatK is plotted relative to the maximum obtained 7 dpi, while the abundance of the RNA precursor (pre-trnK-matK) and mature trnK is plotted relative to the maximum 25 and 59 dpi, respectively (set to 1). Reference parameters of the reaction kinetics of model 1 and model 2 are given in “Materials and Methods.” Note the different scaling.