Whole Cells - Molecular Theory of the Living Cell - page 386

12.3 Simultaneous Measurements of Transcript Levels (TL)
and Transcription Rates (TR) in Budding Yeast
Both the transcript level (TL) and transcription rate (TR) of individual genes were
measured with DNA arrays at the same time by Garcia-Martinez et al. (2004).
The S. cerevisiae yeast strain BQS252 was grown overnight at 28 C in YPD
medium (2% glucose, 2% peptone, 1% yeast extract) to exponential growth
phase. Cells were recovered by centrifugation, resuspended in YPGal medium
(2% galactose, 2% peptone, 1% yeast extract), and allowed to grow in YP Gal
medium for up to about 14 h after the glucose-galactose shift. Cell samples were
taken at 0, 5, 120, 360, 450, and 850 min after the glucose-galactose shift. Two
different aliquots were taken from the cell culture at each sampling time. One
aliquot was processed to measure transcription rates (TR) using the genomic run-on
protocol , the scaled-up version of the usual nuclear run-on method (Hirayoshi and
Lis 1999), and the other was processed to measure transcript levels (TL) using the
same DNA macroarrays recovered from the associated TR measurements. Two
examples of the TL and TR measurements are shown in Fig. 12.2 .
One striking feature of the curves shown in Fig. 12.2 is the contrasting kinetic
behaviors of the glycolytic and oxidative phosphorylation (or oxphos) transcripts in
Panel a and the close similarity of the transcription-rate profiles in Panel b of these
two metabolic pathways. It is important to keep in mind that at least a part of the
reason for this difference is attributable to the fact that Panel a deals with RNA
concentrations whereas Panel b is concerned with the rates of RNA synthesis.
12.4 RNA Trajectories as Intracellular Dissipative
Structures (IDSs) or RNA Dissipatons
The temporal trajectories of individual transcripts (i.e., RNA molecules) shown in
Fig. 12.1 are dynamic structures whose existence depends on dissipating free energy
and hence can be referred to as dissipative structures of Prigogine (1977, 1980)
(see Sect. 3.1 ) . These RNA trajectories (or waves) are also frequently referred to as
“gene expression profiles,” which is an inaccurate statement because RNA
trajectories reflect not only gene expressions (understood here as transcription) but
also transcript degradation (see Step 2 in Fig. 12.4 below). The general decline in the
average RNA levels shown in Fig. 12.1d during the first 360 min after switching
glucose to galactose is primarily due to the depletion of intracellular ATP caused by
the removal of glucose, the preferred energy source of budding yeast. The subsequent
rise in RNA levels beginning around 360 min is most likely due to galactose-induced
expression of the genes coding for the Leloir enzymes (Berg et al. 2002) needed to
metabolize the new substrate, galactose, to generate ATP (Ji et al. 2009a). This
interpretation is supported by the finding that the Leloir transcripts began to increase
at about 120 min after the nutritional shift (see Fig. 12.3 ).