Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription.
In trypanosomes, most genes are arranged in polycistronic transcription units. Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation. Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting.
McAndrew
M
M
Zentrum für Molekulare Biologie, Heidelberg, Im Neuenheimer Feld 282, D-69120, Heidelberg, Germany.
Graham
S
S
Hartmann
C
C
Clayton
C
C
D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D018503:Genome, Protozoan; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014246:Trichomonas vaginalis / Q000235:genetics; D014345:Trypanosoma / Q000235:genetics* / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000096:biosynthesis* / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D008156:Luciferases; D012319:RNA Polymerase II