Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution

Abstract

Transcribing RNA polymerases oscillate between three stable states, two of which, pre- and posttranslocated, were previously subjected to x-ray crystal structure determination. We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or "backtracked" state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the "P" site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS). Structure determination of a cocrystal with TFIIS reveals a rearrangement whereby cleavage of the RNA may take place.

Fig. 1

The three states of a pol II transcription elongation complex. RNA transcript is red, DNA template is blue. The nucleotide base just added to the 3’-end of RNA, and the complementary base in the DNA template, are represented by cyan and green bars. The dashed oval represents the empty nucleotide addition site in the post-translocation state. The green circle represents the pol II bridge helix.

Fig. 2

Structure of pol II elongation complex in the backtracked state. (A) Complex with one mismatched residue at the 3’-end of the RNA (12mer RNA). The view is a standard one, from the “Rpb2 side,” as in the past (22–25). Difference electron density map (F_o-F_c omit map, contoured at 3.0 sigma) between backtracked and post-translocation complexes is shown in green mesh. RNA and DNA are red and cyan. Ribonucleotides at +1 and +2 positions in the RNA are yellow and blue. Parts of bridge helix (Rpb1 825–848) and trigger loop (Rpb1 1070–1100) are green and cyan. Rpb1 T827 is orange. Side chains of Rpb1 Q1078 and N1082 are also shown. Mg²⁺ ion is represented by a magenta sphere. (B) The binding pocket for backtracked ribonucleotide at position +2. View is rotated from that in (A) to better reveal side chain interactions. Rpb1 440–450, Rpb1 470–481, bridge helix (Rpb1 810–848), trigger loop(1070–1100), Rpb2 760–776 are in orange, lime green, green, cyan and hot pink, respectively. Side chains of Rpb1 R446, N479, L824, T827, Q1078, N1082 and Rpb2 Y769 are shown. Other colors as in (A). The binding pocket is highlighted by a dashed green circle. (C) Backtracked RNA is kinked toward the bridge helix and differs from canonical A form RNA. Backbones of backtracked RNA (red, yellow, and blue) and canonical A form RNA (gray) are superimposed. Color scheme as in (A). View is rotated 90° counterclockwise about a vertical axis from that in (A). (D) Comparison of backtracked complex structures with one and two base mismatches. The one-base-mismatch structure is shown as in (A) with the same color code but in backbone representation. The two-base-mismatch structure is shown in magenta. (E) Structure of elongation complex backtracked due to a cisplatin damage site in the template DNA. Graphics same as in (A). (F) Molecular dynamics simulation of bases in the two-base-mismatch (13mer RNA) complex. Deviations of bases two (A10) and one (G12) residues from the 3’-end, and of the 3’-terminal residue (C13), as a function of time.

Fig. 3

Structure of backtracked complex with seven mismatched residues at the 3’-end of the RNA (18mer RNA). (A) Same representation as Fig. 2A, except that ribonucleotide at +3 position is magenta and phosphate group at +4 position is orange. (B) Interactions of pol II with additional backtracked residues. Rpb1 750–754, Rpb2 760–772, and Rpb2 1018–1022 are lime green, hot pink, and marine. Other colors as in Fig. 2A, view as in Fig. 2C. Side chains of Rpb1 K752, Rpb2 Q763, R766, Y769, S1019, and R1020 are shown.

Fig. 4

Structure of backtracked complex with TFIIS. (A) Clash of backtracked RNA with published structure of TFIIS. Backtracked complex with one mismatched base (12mer RNA), depicted as in Fig. 2A, was superposed with TFIIS(1Y1V), with the use of Coot, based on secondary-structure matching (ssm) of RPB1. The tip of domain III of TFIIS is light blue. Side chains of TFIIS residues 290 and 291 are shown. Parts of bridge helix and trigger loop of 1Y1V complex are wheat. (B) Structure of backtracked complex with two mismatched bases (13mer RNA) complexed with TFIIS. Backtracked complex is depicted as in Fig. 2A, except slightly rotated, and with Rpb2 760–776 in hot pink. The tip of domain III of TFIIS is magenta. Corresponding regions from 1Y1V are superimposed, pol II in wheat and TFIIS in light blue.