DNA bending and wrapping around RNA polymerase: a "revolutionary" model describing transcriptional mechanisms

Abstract

A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription.

FIG. 1

Functional domains of the catalytic subunits of multisubunit RNA polymerases, based primarily on experiments with E. coli RNA polymerase. (A) Homology regions within the catalytic subunits that make specific functional interactions. The catalytic center is a modular structure of β and β′ domains. The active-site Mg²⁺ is held by three clustered aspartic acid residues in region d. The a homology region of β′ holds an atom of Zn²⁺. The a and I regions are thought to make up the processivity sliding clamp, because the protein channels for DNA entry and RNA exit are within the a and I regions. The J region (which also binds Zn²⁺), found in most homologues of Rpb2 but not in β, is not shown. “Hybrid” is the RNA-DNA hybrid. The 3′ end of the RNA chain is indicated by a black dot. (B) Bending of template DNA through the RNA polymerase active site. The open complex appears to be about 10 to 12 single-stranded bases. The RNA-DNA hybrid appears to be 8 to 9 bp. See the text for references.

FIG. 2

A three-step model for initiation by E. coli RNA polymerase. The holoenzyme measures 160 Å in its longest dimension. The footprint of holoenzyme on DNA, however, measures over 300 Å, indicating that promoter DNA must wrap around RNA polymerase. Isomerization is a progression of conformational changes in DNA and protein accompanying each step in the reaction pathway. A major change in protein conformation, the closing of the sliding clamp, is thought to occur during formation of closed complex II. This step results in substantial DNA unwinding but not DNA strand separation. Features of the holoenzyme structure include a palm (P), a channel (C), fingers (F), and a thumb (T). Closing of the fingers and thumb forms the sliding clamp for elongation.

FIG. 3

DNA wrapping in the RNA polymerase II preinitiation complex. TBP is in yellow; B, TFIIB (red); F30, TFIIF RAP30 subunit (green); F74-N, TFIIF RAP74 subunit (amino acids 1 to 205) (green); E34, TFIIE 34-kDa subunit (clear blue); Pol II, RNA polymerase II (opaque blue); HIR1, RAP74 dimerization region (amino acids 172 to 205) (129). Models of transcriptional mechanisms can be viewed at http://labcoulombe.usherb.ca.

FIG. 4

Isomerization of transcription complexes. (A) DNA wrapping around RNA polymerase in the preinitiation complex. The diamond shape represents RNA polymerase prior to isomerization. The football shape containing an asterisk represents the isomerized holoenzyme. A DNA bend in the upstream promoter region and another bend around +1 coupled with a tight DNA wrap around RNA polymerase leads to helix unwinding. (B) Comparison of the E. coli RNA polymerase three-step mechanism and the mammalian RNA polymerase II stepwise-assembly model. Proposed similarities between these mechanisms include DNA bending in the upstream promoter region, DNA bending just downstream of +1, wrapping of DNA around RNA polymerase, and isomerization of RNA polymerase and DNA on the pathway to formation of the open complex. R_II, RNA polymerase II (Pol II); R, RNA polymerase; P, promoter DNA; D, TBP; W, wrapped; L, loose; T, taut; O, open; C1, closed complex I; C2, closed complex II; HIR1, a RAP74 dimerization region (amino acids 172 to 205); ∗, conformational change in RNA polymerase. (C) The TBP-TAF_II complex and the TBP-TFIIB-RNA polymerase II-TFIIF-TFIIE complex (lacking TAF_IIs) (R_IIP_W,T in panel B) appear to isomerize promoter DNA into a similar conformation.

FIG. 4

Isomerization of transcription complexes. (A) DNA wrapping around RNA polymerase in the preinitiation complex. The diamond shape represents RNA polymerase prior to isomerization. The football shape containing an asterisk represents the isomerized holoenzyme. A DNA bend in the upstream promoter region and another bend around +1 coupled with a tight DNA wrap around RNA polymerase leads to helix unwinding. (B) Comparison of the E. coli RNA polymerase three-step mechanism and the mammalian RNA polymerase II stepwise-assembly model. Proposed similarities between these mechanisms include DNA bending in the upstream promoter region, DNA bending just downstream of +1, wrapping of DNA around RNA polymerase, and isomerization of RNA polymerase and DNA on the pathway to formation of the open complex. R_II, RNA polymerase II (Pol II); R, RNA polymerase; P, promoter DNA; D, TBP; W, wrapped; L, loose; T, taut; O, open; C1, closed complex I; C2, closed complex II; HIR1, a RAP74 dimerization region (amino acids 172 to 205); ∗, conformational change in RNA polymerase. (C) The TBP-TAF_II complex and the TBP-TFIIB-RNA polymerase II-TFIIF-TFIIE complex (lacking TAF_IIs) (R_IIP_W,T in panel B) appear to isomerize promoter DNA into a similar conformation.

FIG. 4

Isomerization of transcription complexes. (A) DNA wrapping around RNA polymerase in the preinitiation complex. The diamond shape represents RNA polymerase prior to isomerization. The football shape containing an asterisk represents the isomerized holoenzyme. A DNA bend in the upstream promoter region and another bend around +1 coupled with a tight DNA wrap around RNA polymerase leads to helix unwinding. (B) Comparison of the E. coli RNA polymerase three-step mechanism and the mammalian RNA polymerase II stepwise-assembly model. Proposed similarities between these mechanisms include DNA bending in the upstream promoter region, DNA bending just downstream of +1, wrapping of DNA around RNA polymerase, and isomerization of RNA polymerase and DNA on the pathway to formation of the open complex. R_II, RNA polymerase II (Pol II); R, RNA polymerase; P, promoter DNA; D, TBP; W, wrapped; L, loose; T, taut; O, open; C1, closed complex I; C2, closed complex II; HIR1, a RAP74 dimerization region (amino acids 172 to 205); ∗, conformational change in RNA polymerase. (C) The TBP-TAF_II complex and the TBP-TFIIB-RNA polymerase II-TFIIF-TFIIE complex (lacking TAF_IIs) (R_IIP_W,T in panel B) appear to isomerize promoter DNA into a similar conformation.

FIG. 5

A proposed additional step in preinitiation complex assembly involving movement of TAF_IIs from the core promoter region, in order to bind RNA polymerase II and the general transcription factors (GTFs). The promoter is depicted as an isomerized DNA structure tightly wrapped around a TAF_II core, part of which may resemble a core histone octamer (hTAF_II80/70-hTAF_II32/31 heterotetramer-hTAF_II20/15 tetramer). RNA polymerase II and associated general transcription factors dock first in the upstream promoter region through interactions with TBP, TFIIA, promoter DNA, and TAF_IIs. TAF_IIs must be displaced from the core promoter for RNA polymerase II to bend the initiator sequence through the enzyme active site. This step requires some movement of the DNA. Notice the similarities between this mechanism and that proposed for E. coli RNA polymerase (Fig. 2). hTAF_II250 is indicated as an extended structure linking the TAF_II core with TBP. This connection between hTAF_II250 and TBP appears to be missing in yTAF_II145/130.

FIG. 6

Isomerization of the transcription elongation complex. (A) Proposed kinetic pathways for isomerization, phosphodiester bond formation, backtracking, arrest, and editing by RNA polymerases. EC is the unisomerized elongation complex, and EC* is the isomerized elongation complex. TFIIF stabilizes EC* by stimulating k_i(n) and/or inhibiting k_−i(n). TFIIF is not expected to affect k*(n + 1), the rate constant for phosphodiester bond synthesis. Rates for RNA polymerase active-site translocation are not shown in this model. Translocation rates are expected to be rapid relative to the rates shown. TFIIS, GreA, and GreB are expected to act on the EC, helping to resolve backtracked complexes by stimulating an endonuclease activity of their respective RNA polymerase, releasing oligonucleotides such as dinucleotides (N₂). (B) EC* is thought to differ from EC by sharp DNA bending through the RNA polymerase active site and more extensive helix unwinding.

FIG. 7

Models for isomerized RNA polymerase II elongation complexes in the absence (A) and presence (B) of TFIIF. The RAP74 dimerization region HIR1 (amino acids 172 to 205) is critical for accurate initiation (84), elongation stimulation (84), and DNA wrapping in the preinitiation complex (129). The RNA exit channel and DNA entry channel are drawn so that they contact the RNA polymerase sliding clamp (109).