Multisubunit RNA polymerases (RNAP) are responsible for genomic transcription of protein-coding genes in all organisms from bacteria to humans. These RNAP exhibit strong conservation in the core subunit sequence, 3-dimensional structure, and protein-nucleic acid contacts (1
). Despite this conservation, the extent to which evolution has produced functional divergence, especially in surface-located motifs that bear a superficial resemblance to each other, is generally unknown. An excellent case in point is the flap tip in the second-largest subunit of RNAP (β in bacteria; RPB2 in eukaryotes). In prokaryotes, the β flap tip (corresponding to the flap loop on the wall domain in eukaryotic RPB2) is capped by a hydrophobic alpha helix (the flap tip helix) located near the mouth of the RNA exit channel. The flap tip helix helps recruit different regulators during the initiation and elongation phases of transcription (e.g., σ and NusA, respectively) (17
Interaction of the flap tip helix with σ region 4 is essential for transcription initiation at the major class of bacterial promoters that depend on consensus −10 and −35 promoter elements. Deletion of the flap tip helix (ΔFTH) in Escherichia coli
RNAP (Δ887-897 in the E. coli
β subunit, corresponding to Δ873-884 in human RPB2) blocks initiation at −35-dependent promoters but not at extended −10 promoters (17
). Interaction of the flap tip helix with σ region 4 positions σ region 4.2 for contact with the −35 promoter element (17
). Although the interaction of the flap tip with region 4 of the σ factor is essential for initiation, this interaction also hinders promoter escape (35
In E. coli
RNAP, the flap tip also plays roles in transcript elongation. It is required for pause enhancement by nascent RNA hairpins at pause sites (46
). In addition, the flap tip helix is required for enhancement of pausing or termination by the elongation factor NusA (19
). Thus, the flap domain in E. coli
RNAP plays a significant role via direct protein-protein interactions in transcription initiation, as an indirect modulator of active-site properties in pausing, and in protein factor-mediated control of transcriptional pausing at the his
A recent X-ray cocrystal structure of yeast RNAPII with transcription factor IIB (TFIIB) reveals flap loop and flap loop helix structures in RPB2 that are similar in structure and location to the flap tip and flap tip helix in bacterial RNAP (26
) (see A). However, the eukaryotic flap loop exhibits only limited sequence similarity to the bacterial flap tip. In the TFIIB-RNAPII structure, the flap loop helix contacts the N-terminal ribbon domain of TFIIB, which may be analogous to the interaction of region 4 of σ with the flap tip helix of bacterial RNAP (26
). The TFIIB reader segment lies C-terminal of the TFIIB ribbon domain and appears to make key contacts with a promoter element involved in start site selection (26
). These similarities between the flap tip and flap loop of bacterial and eukaryotic RNAP, respectively, raise the possibility that the flap loop could play key roles in transcription initiation and promoter escape by eukaryotic RNAPII.
Fig. 1. Expression of wild-type and ΔFL ettRNAPII. (A) Structure of Saccharomyces cerevisiae RNAPII in complex with TFIIB (Protein Data Bank identification code [PDB ID] 3k1f) (26) with the flap loop (blue/green) enlarged in the inset. The flap loop deletion (more ...)
Transcript elongation in eukaryotes is regulated by multiple protein factors that affect transcript elongation by RNAPII either positively or negatively but whose precise contacts with RNAPII are unknown (e.g., the positive factor TFIIF and the negative factor DSIF/NELF, which is composed of negative elongation factor [NELF] and 5,6-dichloro-1-β-d
-ribofuranosylbenzimidazole [DRB] sensitivity-inducing factor [DSIF]) (15
). Recent studies using cross-linking–mass spectrometry (4
) and protein footprinting (14
) suggest that the TFIIF dimerization domain contacts the lobe domain of RNAPII close to RPB9 and place the Tfg2/RAP30 subunit of TFIIF near the wall domain of RNAPII. This mapping raises the possibility that Tfg2/RAP30, which bears some similarity to bacterial σ, contacts the flap loop, a contact analogous to that of the bacterial elongation factor NusA with the flap tip. NELF, in complex with DSIF, induces promoter-proximal transcriptional pausing (38
), in part by interacting with the emerging nascent RNA (51
). The flap loop, which is located at the mouth of the RNA exit channel, could facilitate the interaction of NELF with the emerging transcript.
During transcript elongation, at certain sequences on the DNA, RNAPII reverse translocates, or backtracks, along RNA and DNA chains with the 3′ end of the RNA dislodged from the active site and located in the secondary channel (20
). Interaction of the exiting RNA with the flap loop located immediately outside the RNA exit channel might provide a physical barrier to backtracking and might thus play a key role in maintaining the active state of the enzyme.
Based on these similarities, we sought to test whether the flap loop of human RNAPII plays a significant role in transcription by RNAPII. To study the role of the flap loop in vivo and in vitro, we constructed and studied the effects of a deletion of the flap loop (ΔFL; RPB2 Δ873-884). We used immunoprecipitation (IP) of formaldehyde-cross-linked DNA with monoclonal antibodies (MAbs) specific for the mutant RNAPII followed by hybridization to promoter region tiling arrays (chromatin IP [ChIP]-chip) to study the activity of RNAPII lacking the flap loop in human cell lines. We also purified the mutant human RNAPII from the same stable cell lines and used in vitro transcription assays to test for essential roles of the flap loop in either transcription initiation or elongation.