The use of a synthetic tRNA gene as a novel approach to study in vivo transcription and chromatin structure in yeast.
To monitor in vivo transcription and chromatin structure of yeast tRNA genes, we constructed a synthetic tRNA gene that can be used as a reporter. Constructs in which this synthetic tRNA gene is combined with different flanking regions can be integrated into the genome as single copies. The artificial tRNA gene is tagged by the insertion of an intron-like sequence that cannot be spliced out from the precursor and transcripts can thus be identified and quantitated. By several criteria, the artificial tRNA gene behaves like a resident tRNA gene. By measuring the accessibility towards DNaseI in chromatin, we found that the artificial tRNA gene exhibits the same characteristic pattern as resident tRNA genes. Three DNaseI-sensitive sites across the transcribed part of the gene and the immediate flanking regions reflect the formation of the stable transcription complex; positioned nucleosomes are observed in the upstream flanking region. We are confident that the system we have established will prove useful for studying regulatory aspects of tRNA gene expression as well as aspects of pre-tRNA processing and splicing.
Krieg
R
R
Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Universität München, FRG.
Stucka
R
R
Clark
S
S
Feldmann
H
H
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002843:Chromatin / Q000648:ultrastructure*; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal; D012343:RNA, Transfer / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014981:Xenopus / Q000235:genetics
D002843:Chromatin; D004271:DNA, Fungal; D012322:RNA Precursors; D012331:RNA, Fungal; D012343:RNA, Transfer