Wg. Gu et al., NASCENT RNA CLEAVAGE BY ARRESTED RNA POLYMERASE-II DOES NOT REQUIRE UPSTREAM TRANSLOCATION OF THE ELONGATION COMPLEX ON DNA, The Journal of biological chemistry, 268(34), 1993, pp. 25604-25616
Obstacles incurred by RNA polymerase II during primary transcript synt
hesis have been identified in vivo and in vitro. Transcription past th
ese impediments requires SII, an RNA polymerase II-binding protein. SI
I also activates a nuclease in arrested elongation complexes and this
nascent RNA shortening precedes transcriptional readthrough. Here we s
how that in the presence of SII and nucleotides, transcript cleavage i
s detected during SII-dependent elongation but not during SII-independ
ent transcription. Thus, under typical transcription conditions, SII i
s necessary but insufficient to activate RNA cleavage. RNA cleavage co
uld serve to move RNA polymerase II away from the transcriptional impe
diment and/or permit RNA polymerase II multiple attempts at RNA elonga
tion. By mapping the positions of the 3'-ends of RNAs and the elongati
on complex on DNA, we demonstrate that upstream movement of RNA polyme
rase II is not required for limited RNA shortening (seven to nine nucl
eotides) and reactivation of an arrested complex. Arrested complexes b
ecome elongation competent after removal of no more than nine nucleoti
des from the nascent RNA's 3'-end. Further cleavage of nascent RNA, ho
wever, does result in ''backward'' translocation of the enzyme. We als
o show that one round of RNA cleavage is insufficient for full readthr
ough at an arrest site, consistent with a previously suggested mechani
sm of SII action.