Volume 10, Issue 3, Pages 623-634 (September 2002) Swing-Gate Model of Nucleotide Entry into the RNA Polymerase Active Center  Vitaliy Epshtein, Arkady Mustaev, Vadim Markovtsov, Oxana Bereshchenko, Vadim Nikiforov, Alex Goldfarb  Molecular Cell  Volume 10, Issue 3, Pages 623-634 (September 2002) DOI: 10.1016/S1097-2765(02)00640-8

Figure 1 Transcription Cycle and the Proposed Swing-Gate Mechanism of Nucleotide Entry Elements of protein structure are represented by cylinders, and the nucleic acid scaffold in the ternary complex is depicted by bars. The scaffold is shown according to Korzheva et al. (2000), with one unpaired template base. RNA (pink) base-paired to the DNA template (yellow, with bases 1, 2, and 3 numbered) translocates relative to the active center (symbolized by Mg2+ flanked by the i and i+1 sites). The β′ subunit F bridge (green), which is supported by the helixes of the G loop (white), separates the main channel for the DNA duplex (yellow:purple) from the secondary channel for nucleotides, backtracking, or abortive RNA release. The crucial amino acids, which are evolutionarily conserved, are β′ subunit Lys789 (red), Met932 (blue), Thr790, and Ala791 (cyan) (the numbering is that of E. coli, the crucial residues Lys789 and Met932 are substituted by Arg and Leu in Taq and yeast RNAPs, respectively). (A) The transcription complex before the phosphodiester bond formation. The F bridge is relaxed. (B) The pretranslocation stage. The pink arrow represents the trajectory for RNA during backtracking or abortive release. (C) Translocation. The F bridge bends, causing Lys789 to enter the i+1 site. The bending is induced by Met932 swapping contacts with Lys789. Side chains 790 and 791 clash with the DNA base in the i+1 site, inducing its translocation. (D) Relaxation. The F bridge returns to the straight helix conformation, vacating the i+1 site for the next nucleotide (pink arrow) or for reverse translocation. Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 2 RNA-Protein Contacts in the Transcriptional Complexes of the Wild-Type and Substitution Mutant RNAPs Two types of crosslinking reagents (I and II, with double-pointed arrows showing the length of the reactive groups at the 3′ terminal ribose) were used in the context of the initiating complex Rif-GCU or the elongating complex TEC21. Rif represents Rifampicin covalently linked to trinucleotide G[32P]CU (Mustaev et al., 1994). In TEC21, a 21-meric transcript formed on the T7A1 promoter was labeled with 32P at several cytidine nucleotide positions. The panels show autoradiograms of 4% SDS-PAGE gels with the β and β′ subunits carrying the [32P]RNA adducts. βins corresponds to the β subunit with an insertion into the chromosomal rpoB gene (Mustaev et al., 1991). Under each panel, individual amino acids and regions mapped are indicated. In case of multiple crosslinking in the same subunit, “>” indicates the relative intensity of the crosslinks. Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 3 Mapping of RNA-Protein Contacts in the Initiating Complex (A) Single-hit CNBr degradation patterns of the β′ subunit crosslinked by reagent II (lanes 2–5) or I (lanes 6–7). The reference CNBr degradation pattern (lane 1) was obtained using the subunit labeled C-terminally by 32P at an engineered protein kinase site (marker 2 [M2]). Numbers indicate the positions of Met residues in the E. coli sequence, which correspond to the cleavage products. Enzymes used in the crosslinking experiments (wild-type [WT] or β′ M932L) are indicated. Solid lines in the autoradiograph connect the identical cleavage products. Dotted lines indicate the putative position of the next expected cleavage product, which is not radioactive. (B) NTCBA degradation products of the β′ subunit crosslinked by reagent II (lanes 2 and 4) and reagent I (lane 3). Reference degradation patterns of the C-terminally labeled (as above, M3) and N-terminally labeled (marker 4 [M4]; taken from Korzheva et al., 2000) subunit are represented at lanes 1 and 5, respectively. (C) Histograms for the intensity of the degradation products for the indicated lanes of Figure 2A. Corresponding products are numbered. (D) The products of exhaustive CNBr degradation of the β′ subunit crosslinked in initiation complex. The position and the molecular weight of the markers are indicated. (E) I: the position of the crosslinked adduct and of Cys residues in the 822–1026 segment of β′ subunit. II: the single-hit NTCBA degradation pattern of the faster migrating product from (D). (F) CNBr cleavage of the β subunit crosslinked as in (A). M1 (marker 1) represents the reference degradation pattern obtained from the same subunit crosslinked near the COOH terminus, according to (Korzheva et al., 2000). (G) Histograms of the CNBr degradation products from (F). Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 4 Mapping of RNA-Protein Contacts in Elongating Complexes (TEC 21) (A) Single-hit NTCBA degradation patterns of the β′ subunit crosslinked by reagent I (lane 2) and by reagent II (lane 3) along with the reference patterns originated from the subunit labeled near the COOH (lane 1, M2) or NH2 (lane 4, M3) termini. (B) Single-hit CNBr degradation patterns of the β′ subunit crosslinked by reagent I (lanes 2 and 3) and reagent II (lanes 4 and 5). The reference C-terminal CNBr degradation pattern was obtained as above (M2, lane 1). (C) Intensities of CNBr degradation products from (B) revealed by phosphoimaging. (D) Normalized intensities of the same degradation products. (E) Single-hit CNBr degradation patterns of the crosslinked β subunit crosslinked by reagent I (lanes 2 and 3) and by reagent II (lanes 4 and 5). Marker 1 (M1) is the same as in Figure 3F. (F) Intensities of the CNBr degradation products from (E). Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 5 Projections of the Contacts of the 3′ Terminus on RNAP Structural Models in Space-Filled and Ribbon Renditions Color codes are the same as in Figure 1. The bent conformation (A and B) is from the structural model of the Taq ternary complex (Korzheva et al., 2000). The straight conformation (C and D) is from the crystal structure of the yeast Pol II ternary complex (Gnatt et al., 2001). Note that the terminal base pair in the bent conformation (A and B) is in the i site, whereas in the straight conformation (C and D), it is in the i+1 site. The space filled models (A and C) are supplemented with the arbitrary drawn unstructured “G loop” (white circles) and show the β Lys1065 residue (red, top right corner). In (C), three nucleotides of the 3′ terminus of RNA that has entered the secondary channel are shown in cyan to illustrate the crosslink with Met932. Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 6 Comparison of the T934A Substitution Mutant with the Wild-Type RNAP (A) Phosphoimager scans of autoradiograms of Figure 2, lanes 1 and 4, representing crosslinking of reagent I to the wild-type (WT) and the mutant RNAP, respectively. The two peaks correspond to the β and β′ subunits. (B) Products of runoff transcription assays on the T7A1 promoter fragment at different NTP concentrations. (C) Fe2+-mediated cleavage of RNA in TEC20, TEC26, and TEC32. Autoradiograms of the RNA products show the effect of incubation of the TECs with Fe(NH4)2(SO4)2. (D) Pyrophosphorolysis of RNA. Autoradiograms of the RNA products show the effect of incubation of TEC20 with PPi. Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)

Figure 7 Principal Interactions in the Swing-Gate Structure Taq Core ([A], bent), T. thermophilus holoenzyme ([B], intermediate), and yeast Pol II ([C], straight) RNA polymerases. The active center Mg is shown as a cyan sphere. Amino acid residues involved in the principal interactions are shown in space-filled representation and are numbered. Lys789 and Met932 are highlighted in red and magenta, respectively. Molecular Cell 2002 10, 623-634DOI: (10.1016/S1097-2765(02)00640-8)