Transcriptional
initiation under conditions of anoxia-induced quiescence in mitochondria
from Artemia franciscana embryos
B.D.
Eads, S.C. Hand-2003
Journal
of Experimental Biology, 206(3): 577-589
(from ISI Current Contents)
Abstract:
In response to anoxia, embryos of the brine shrimp Artemia franciscana are
able coordinately to downregulate metabolism to levels low enough to permit
survival for several years at room temperature. In addition to dramatic
decreases in free ATP levels and heat production, intracellular pH drops
from 7.8 to 6.3 overnight. Use of isolated mitochondria to study
transcriptional responses to anoxia offers several advantages: (1) the
localized nature of transcript initiation, processing and degradation, all
of which may be followed in organello; (2) the relatively simple cis- and
trans-machinery involved and (3) the ability to provide relevant
physiological treatments in vitro. In response to anoxic incubation of
embryos in vivo for 4h followed by anoxic mitochondrial isolation and anoxic
transcription assay at pH 6.4, a significant decrease in overall UTP
incorporation (77%) was seen after 30min relative to normoxic, pH 7.9
controls. A less severe inhibition of transcription under anoxia (52%) was
observed compared with controls when pH was raised to 7.9. Similarly, under
normoxia, the incubation at low pH (6.4) reduced transcription by 59%.
Ribonuclease protection assays showed that the contribution of vitro
initiation during the assay fell from 78% at pH 7.9 to approximately 32% at
pH 6.4 under either normoxic or anoxic conditions. DNA footprinting of
putative transcriptional promoters revealed proteins at regular intervals
upstream of the 12S rRNA in the control region, which previously had been
indirectly inferred to contain promoters for H-strand transcription. The
area between 12 030 and 12 065 contains a sequence in the tRNA(leu) gene
believed to bind the transcription termination factor mTERF or TERM, and we
provide the first evidence that this sequence is protein-bound in A.
franciscana. However, our hypothesis that initiation is reduced at low pH
because of a change in DNA binding by mitochondrial transcription factors
was not confirmed. We propose that regulation of initiation may be mediated
by covalent modification or by protein-protein interactions not detected by
footprinting.
(Louisiana State Univ., Dept. Sci. Biol., Baton
Rouge, LA 70803, USA, e-mail of S.C. Hand: shand@lsu.edu)