PDBsum entry 1i6h

Transcription/DNA-RNA hybrid

PDB id: 1i6h

Contents

Protein chains

1381 a.a. *

1097 a.a. *

266 a.a. *

214 a.a. *

84 a.a. *

133 a.a. *

119 a.a. *

65 a.a. *

114 a.a. *

46 a.a. *

DNA/RNA

Metals

_MG

_ZN ×8

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis of transcription: an RNA polymerase ii elongation complex at 3.3 a resolution.

Authors

A.L.Gnatt, P.Cramer, J.Fu, D.A.Bushnell, R.D.Kornberg.

Ref.

Science, 2001, 292, 1876-1882. [DOI no: 10.1126/science.1059495]

PubMed id

11313499

Abstract

The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.

Figure 5.
Fig. 5. DNA-RNA hybrid conformation. The view is similar to that in Fig. 2C. The conformation of the DNA-RNA hybrid is intermediary between canonical A- and B-DNA. DNA, blue; RNA, red.

Figure 6.
Fig. 6. Proposed transcription cycle and translocation mechanism. (A) Schematic representation of the nucleotide addition cycle. The nucleotide triphosphate (NTP) fills the open substrate site (top) and forms a phosphodiester bond at the active site ("Synthesis"). This results in the state of the transcribing complex seen in the crystal structure (middle). We speculate that "Translocation" of the nucleic acids with respect to the active site (marked by a pink dot for metal A) involves a change of the bridge helix from a straight (silver circle) to a bent conformation (violet circle, bottom). Relaxation of the bridge helix back to a straight conformation without movement of the nucleic acids would result in an open substrate site one nucleotide downstream and would complete the cycle. (B) Different conformations of the bridge helix in pol II and bacterial RNA polymerase structures. The view is the same as in Fig. 2C. The bacterial RNA polymerase structure (2) was superimposed on the pol II transcribing complex by fitting residues around the active site. The resulting fit of the bridge helices of pol II (silver) and the bacterial polymerase (violet) is shown. The bend in the bridge helix in the bacterial polymerase structure causes a clash of amino acid side chains (extending from the backbone shown here) with the hybrid base pair at position +1.

The above figures are reprinted by permission from the AAAs: Science (2001, 292, 1876-1882) copyright 2001.

Secondary reference #1

Title

Structural basis of transcription: RNA polymerase ii at 2.8 angstrom resolution.

Authors

P.Cramer, D.A.Bushnell, R.D.Kornberg.

Ref.

Science, 2001, 292, 1863-1876. [DOI no: 10.1126/science.1059493]

PubMed id

11313498

Figure 7.
Fig. 7. Four mobile modules of the Pol II structure. (A) Backbone traces of the core, jaw-lobe, clamp, and shelf modules of the form 1 structure, shown in gray, blue, yellow, and pink, respectively. (B) Changes in the position of the jaw-lobe, clamp, and shelf modules between form 1 (colored) and form 2 structures (gray). The arrows indicate the direction of charges from form 1 to form 2. The core modules in the two crystal forms were superimposed and then omitted for clarity. (C) The view in (B) rotated 90° about a vertical axis. The core and jaw-lobe modules are omitted for clarity. In form 2, the clamp has swung to the left, opening a wider gap between its edge and the wall located further to the right (not shown).

Figure 8.
Fig. 8. Active center. Stereoview from the Rpb2 side toward the clamp. Two metal ions are revealed in a [A]-weighted mF[obs] DF[calc] difference Fourier map (shown for metal B in green, contoured at 3.0 ) and in a Mn2+ anomalous difference Fourier map (shown for metal A in blue, contoured at 4.0 ). This figure was prepared with BOBSCRIPT (85) and MOLSCRIPT (86).

The above figures are reproduced from the cited reference with permission from the AAAs

Secondary reference #2

Title

Architecture of RNA polymerase ii and implications for the transcription mechanism.

Authors

P.Cramer, D.A.Bushnell, J.Fu, A.L.Gnatt, B.Maier-Davis, N.E.Thompson, R.R.Burgess, A.M.Edwards, P.R.David, R.D.Kornberg.

Ref.

Science, 2000, 288, 640-649. [DOI no: 10.1126/science.288.5466.640]

PubMed id

10784442

Figure 4.
Fig. 4. Jaws. (A) Stereoview of structural elements constituting the jaws (left) and the location of these elements within pol II (right). (B) Mobility of the larger, NH[2]-terminal domain of Rpb5. Backbone models of free Rpb5 [gray (47)] and Rpb5 in pol II (pink) are shown with their smaller, COOH-terminal domains superimposed. (C) Conservation of amino acid residues of Rpb5.

Figure 6.
Fig. 6. Topology of the polymerizing complex, and location of Rpb4 and Rpb7. (A) Nucleic acid configuration in polymerizing (top) and backtracking (bottom) complexes. (B) Structural features of functional significance and their location with respect to the nucleic acids. A surface representation of pol II is shown as viewed from the top in Fig. 3. To the surface representation has been added the DNA-RNA hybrid, modeled as nine base pairs of canonical A-DNA (DNA template strand, blue; RNA, red), positioned such that the growing (3') end of the RNA is adjacent to the active site metal and clashes with the protein are avoided. The exact orientation of the hybrid remains to be determined. The nontemplate strand of the DNA within the transcription bubble, single-stranded RNA and the upstream DNA duplex are not shown. (C) Cutaway view with schematic of DNA (blue) and with the helical axis of the DNA-RNA hybrid indicated (dashed white line). An opening in the floor of the cleft that binds nucleic acid exposes the DNA-RNA hybrid (pore 1) to the inverted funnel-shaped cavity below. The plane of section is indicated by a line in (B), and the direction of view perpendicular to this plane (side) is as in Fig. 3. (D) Surface representation as in (B), with direction of view as in (C). The molecular envelope of pol II determined by electron microscopy of 2D crystals at 16 Å resolution is indicated (yellow line), as is the location of subunits Rpb4 and Rpb7 (arrow, Rpb4/7), determined by difference 2D crystallography (25).

The above figures are reproduced from the cited reference with permission from the AAAs