Transcriptional Regulation pp 593-607
Detection and Characterization of Transcription Termination
In most eukaryotes, the generation of the 3′ end and transcription termination are initiated by cleavage of the pre-mRNA upstream of the polyadenylation site. This cleavage initiates 5′–3′ degradation of the 3′ end cleavage product by the exoribonuclease Rat1p leading to the dissociation of the RNA polymerase II (RNAPII) complex. The Rat1p-dependent transcription termination was also shown to be initiated by a polyadenylation-independent cleavage performed by the double-stranded RNA-specific ribonuclease (RNase) III (Rnt1p) suggesting that the majority of transcription termination events are RNase dependent. Therefore, it became essential for future studies on transcription termination to carefully consider both the nature of the RNase-dependent RNA transcripts and the association pattern of the RNAPII with the transcriptional unit. Here, we present methods allowing the evaluation of the impact of yeast RNases on the 3′ end formation and their contribution to transcription termination. Northern blot analysis of transcripts generated downstream of known genes in the absence of RNases identifies potential transcription termination sites while chromatin immunoprecipitation of RNAPII differentiates between termination- and transcription-independent processing events.
Key wordsRNase III Rat1p Rnt1p Termination ChIP Northern blot Ribonucleases