Interaction of RNA Polymerase II Fork Loop 2 with Downstream Non-template DNA Regulates Transcription Elongation

Zachary F. Burton,Céline Domecq,Mikhail Kashlev,Benoit Coulombe,Maria L. Kireeva

doi:10.1074/jbc.m111.260844

Abstract

Fork loop 2 is a small semiconservative segment of the larger fork domain in the second largest Rpb2 subunit of RNA polymerase II (Pol II). This flexible loop, juxtaposed at the leading edge of transcription bubble, has been proposed to participate in DNA strand separation, translocation along DNA, and NTP loading to Pol II during elongation. Here we show that the Rpb2 mutant carrying a deletion of the flexible part of the loop is not lethal in yeast. The mutation exhibits no defects in DNA melting and translocation in vitro but confers a moderate decrease of the catalytic activity of the enzyme caused by the impaired sequestration of the NTP substrate in the active center prior to catalysis. In the structural model of the Pol II elongation complex, fork loop 2 directly interacts with an unpaired DNA residue in the non-template DNA strand one nucleotide ahead from the active center (the i+2 position). We showed that elimination of this putative interaction by replacement of the i+2 residue with an abasic site inhibits Pol II activity to the same degree as the deletion of fork loop 2. This replacement has no detectable effect on the activity of the mutant enzyme. We provide direct evidence that interaction of fork loop 2 with the non-template DNA strand facilitates NTP sequestration through interaction with the adjacent segment of the fork domain involved in the active center of Pol II.

Highlights

Rpb2 Fork Loop 2 in Transcription Elongation stranded DNA template [13], suggesting that the role of fork loop 2 requires further investigation
In the crystal structures of ternary elongation complex (TEC) by T. thermophilus RNA polymerase (RNAP) the loop is juxtaposed against the iϩ2 base pair of the downstream DNA [4, 5]
Due to its particular location, several groups have proposed that the fork loop participates in DNA melting and forward translocation of the enzyme [5,6,7, 10]

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—The wild type Pol II and its variants carrying mutations in the Rpb subunit were purified as described [13]. In the typical assembly reaction, 5–10 pmol of Pol II was incubated with 15–30 pmol of the preannealed RNADNA hybrid in the total volume of 30 –50 ␮l for 10 min at room temperature; 30 –100 pmol of non-template DNA strand were subsequently added for another 10 min. Transcription assays in time intervals shorter than 5 s were performed using the RQF-3 or RQF-4 instruments (Kintek Corp., Austin, TX) In this case the reactions were stopped by addition of 1 M HCl or 0.5 M EDTA, and the samples were processed as described previously [22]. The TECs were assembled with the 5Ј-end labeled nontemplate or template DNA strand oligonucleotide. The template DNA strand oligonucleotide was gel-purified after the labeling. Exonuclease III (Exo III) Footprinting [25]—The TECs were assembled with a 5Ј-end labeled template DNA strand. The DNA products were resolved in a 10% acrylamide gel in denaturing conditions

RESULTS

WT dFL

DISCUSSION