RNA chain elongation and termination by mammalian RNA polymerase III. Analysis of tRNA gene transcription by imposing a reversible factor-mediated block to elongation using a sequence-specific DNA binding protein.
We have used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3' end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle. lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.
Syroid
D E
DE
McMaster University Health Sciences Centre, Department of Biochemistry, Hamilton, Ontario, Canada.
Capone
J P
JP
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D012320:RNA Polymerase III / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D012343:RNA, Transfer; D012320:RNA Polymerase III