Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei

Nucleic Acids Res. 1998 Oct 15;26(20):4626-34. doi: 10.1093/nar/26.20.4626.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacteriophage T7 / enzymology*
  • Chromatin / genetics*
  • DNA, Protozoan / genetics
  • DNA, Ribosomal / genetics
  • DNA-Directed RNA Polymerases / metabolism*
  • Doxycycline / pharmacology
  • Gene Expression Regulation
  • Genes, Reporter
  • Mice
  • Operator Regions, Genetic / genetics
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • Promoter Regions, Genetic / genetics
  • Proteins / genetics
  • RNA, Antisense
  • Terminator Regions, Genetic / genetics
  • Tetracycline / pharmacology
  • Transcription, Genetic* / drug effects
  • Transfection
  • Trypanosoma brucei brucei / genetics*
  • Trypanosoma brucei brucei / pathogenicity
  • Trypanosomiasis, African / parasitology
  • Viral Proteins

Substances

  • Chromatin
  • DNA, Protozoan
  • DNA, Ribosomal
  • Proteins
  • RNA, Antisense
  • Viral Proteins
  • Parp1 protein, mouse
  • Poly (ADP-Ribose) Polymerase-1
  • Poly(ADP-ribose) Polymerases
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Tetracycline
  • Doxycycline