Eb. Rasmussen et Jt. Lis, SHORT TRANSCRIPTS OF THE TERNARY COMPLEX PROVIDE INSIGHT INTO RNA-POLYMERASE-II ELONGATIONAL PAUSING, Journal of Molecular Biology, 252(5), 1995, pp. 522-535
Expression of the hsp70 gene of Drosophila melanogaster is controlled
at the level of transcript elongation. In the uninduced state, hsp70 p
ossesses an RNA polymerase II complex, elongationally engaged, but pau
sed early in the transcription unit. In this study, we have used a pow
erful new selection-amplification technique to analyze the RNA transcr
ipts associated with such ''paused polymerases'' under non-heat shock
and heat shock-induced conditions. They reveal a region of pausing on
the uninduced gene in vivo spanning from +21 to +35. This region is in
terrupted by an area of low polymerase density, centered at about +26.
Upon induction, an accumulation of short transcripts, similar in size
to those associated with the paused complex, was seen. Models for pol
ymerase pausing and release are discussed in light of these data. The
increased sensitivity of our new technique also allowed us to investig
ate ternary complexes associated with genes with much lower levels of
engaged RNA polymerase. These included two of the small heat shock gen
es (hsp26 and hsp27) and two metabolic genes (Gapdh-1 and Gapdh-2), wh
ere paused polymerases were thought to be present, plus two other gene
s (Mtn and yp1), where polymerase pausing has not been detected in the
past. In both of the small heat shock genes, we found evidence of pre
viously unknown sites of transcriptional termination, residing immedia
tely upstream of the regions of polymerase pausing. (C) 1995 Academic
Press Limited