Nascent RNA cleavage by arrested RNA polymerase II does not require upstream translocation of the elongation complex on DNA.
Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII-dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in "backward" translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.
Gu
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Graduate Program in Biochemistry & Molecular Biology, Emory University, Atlanta, Georgia.
Powell
W
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Mote
J
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Reines
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D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006657:Histones / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012313:RNA / Q000096:biosynthesis / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D015183:Restriction Mapping; D013698:Templates, Genetic; D014158:Transcription, Genetic
D006657:Histones; D012313:RNA; D004247:DNA; D012319:RNA Polymerase II; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III