Nucleic Acids Research, Vol 26, Issue 20 4626-4634, Copyright © 1998 by Oxford University Press
E Wirtz, M Hoek and GA Cross
Inability of T7 RNA polymerase to processively transcribe higher eukaryotic
chromatin is interpreted as a correlate of its reported inhibition by
nucleosomes on reconstituted templates in vitro . We used chromosomally
integrated reporter cassettes to examine features of T7 transcription in a
lower eukaryotic system. Luciferase reporters were targeted to rDNA in
transgenic Trypanosoma brucei stably expressing the phage polymerase.
Because trypanosome mRNAs are capped by RNA splicing in trans , T7
transcription could be gauged by luciferase activity. In contrast to
findings from higher eukaryotes, T7 transcription is vigorous and
processive on chromatin templates in T.brucei , surpassing levels achieved
with endogenous promoters, including those recruiting RNA polymerase I.
This may be a reflection of intrinsic differences in chromatin structure
between differently evolved eukaryotes or of an integration site that is
exceptionally permissive for T7 transcription due to a local accessible
chromatin conformation. T7 transcription could be manipulated to achieve
different levels of constitutive expression, through the use of promoter
mutations. Moreover, T7 initiation could be regulated by the prokaryotic
Tet repressor and elongation halted by T7 terminator sequences. We have
exploited these features to construct a robust inducible expression system,
whose utility potentially extends to other trans -splicing organisms.
ARTICLES
Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei
Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA.
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