RNA polymerase II transcription: structure and mechanism

Abstract

A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

Fig. 1. Sequence alignments of the TFIIB finger and linker regions

Primary sequences of TFIIB/TFB proteins from Homo sapiens (TFIIB_Human), Drosophila melanogaster (TFIIB_Drome), Caenorhabditis elegans (TFIIB_Caeel), Saccharomyces cerevisiae (TFIIB_Yeast), Methanococcus thermolithotrophicus (TFB_MT) and Sulfolobus shibatae (TFB_SS) were aligned with the use of ClustalW2 and manually edited. Aligned sequences were highlighted with BOXSHADE. Cartoons representing the secondary structures are drawn based on the 3.8 Å pol II–TFIIB crystal structure [9] and are shown above the sequences in red. The B-finger / B-reader regions of the other two pol II–TFIIB crystal structures determined at 4.3Å [10] and 4.5Å [8] are also shown in purple and cyan. A fragment implicated in binding the initiating nucleotide is indicated by double green lines under the sequence alignment. A tryptophan-containing fragment in TFB located close to the upstream edge of the transcription bubble is indicated by double blue lines.

Fig. 2. Schematic alignment of TFIIB and factor structures

TFIIB and σ are shown in red and green. Corresponding domains of the two proteins are indicated.

Fig. 3. Structural model of the “minimal” closed promoter complex

(A) “Top” view [9] of the closed promoter complex. Pol II, TFIIB, TBP and promoter DNA are all shown in surface representation. The pol II clamp is shown in gold, dock in lime, wall in blue, protrusion in wheat and the rest of pol II in gray. TFIIB is red and TBP is violet. The template and non-template stands are cyan and green respectively. (B) Same as (A) rotated approximately 45 around the y-axis. DNA registers are also indicated. All figures are rendered in PyMOL.

Fig. 4. Tunnels / channels in the pol II–TFIIB complex

Proteins and DNA are colored as in Fig 3, except that pol II and DNA are shown as ribbons, TFIIB and TBP are shown as meshes, and the catalytic magnesium ion is shown as a magenta sphere. View is that of Fig. 2B rotated 45° around the x-axis. Tunnels / channels were calculated using the program MOLE [89] and are shown as semi-transparent surfaces. The template strand tunnel is shown in cyan, the downstream duplex DNA channel in blue and the ribonucleotide entry tunnel in green.

Fig. 5. The multiple states of pol II transcribing complexes

DNA and RNA strands are in cyan and red, with the coding base in the DNA highlighted in blue and the matched base in the RNA strand in purple. The polymerase is symbolized by a gray rectangle and the bridge helix of the polymerase by a green disk. The solid magenta circle represents the catalytic magnesium ion.

Fig. 6. The trigger loop and its interaction with substrate NTP

(A) Structure of the active center region of transcribing complexes with (trigger loop shown in purple) or without (trigger loops shown in in red, blue, or yellow) nucleoside triphosphate (orange). RNA is red, DNA is cyan, magnesium ions are depicted as purple spheres. (B) Expanded view of structure in (A) with NTP bound and with trigger loop in purple. Interactions of trigger loop with NTP are indicated by dashed yellow lines. In addition to the trigger loop, other interacting residues of pol II are shown in yellow and purple. Arrows indicate the flow of electrons during nucleophillic attack of the 3’- OH of the RNA chain terminus upon the α-phosphate of the NTP, for phosphodiester bond formation, phosphoanhydride bond breakage, and pyrophosphate release.

Fig. 7. The bridge helix in straight and bent states

Structures of nucleic acids and the bridge helix in the vicinity of the active center of (A) pol II and (B) RNAP are shown, with the DNA in cyan, the RNA in red, and the bridge helix in purple.

Fig. 8. The structural basis of transcript release

Structure of the RNA-DNA hybrid helix and interacting protein loops in a post-translocation complex.