The fundamental mechanism of transcription is conserved among cellular RNA polymerases. Common features include an unwound region, or “transcription bubble,” of about 15 base pairs of the DNA template and some eight residues of the RNA transcript hybridized with the DNA in the center of the bubble. The enzymes involved, bacterial RNA polymerase (RNAP) and eukaryotic RNA polymerases (pol I, II, and III), are capable of both forward and retrograde movement (“backtracking”) on the DNA. Forward movement is favored by the binding of nucleoside triphosphates (NTPs), while backtracking occurs especially when the enzyme encounters an impediment, such as damaged DNA.
Structures of transcribing complexes have been modeled on the basis of protein-DNA cross-linking data for RNAP and determined by X-ray crystallography for pol II. The first pol II transcribing complex for X-ray crystallography was produced by initiation on a “tailed” template and stalling due to the omission of an NTP (Gnatt et al., 2001
). The resulting structure revealed the complex in the “pre-translocation” state, with the nucleotide just added to the RNA transcript still occupying the addition or “A” site. A more facile and versatile method was subsequently developed, based on the finding that pol II binds a transcription bubble formed from DNA and RNA oligonucleotides and is able to extend the RNA (Kireeva et al., 2000
; Westover et al., 2004
). An X-ray structure obtained by this approach revealed the transcribing complex in the “post-translocation” state, with the A site available for entry of a matched NTP. Crystals of the transcribing complex in the post-translocation state were soaked with a matched nucleotide, UTP, and with an unmatched nucleotide, ATP, revealing a second NTP-binding site, termed the entry or “E” site () (Westover et al., 2004
). All NTPs can bind the E site, whereas only an NTP matched for base pairing with the DNA template binds the A site for addition to the growing RNA chain (Westover et al., 2004
). The addition step is presumed to involve two Mg2+
ions, one stably associated with the enzyme and the other only transiently, entering with the NTP and leaving upon pyrophosphate release.
A and E Sites in the Pol II Transcribing Complex (A)
Beyond these findings, little is known of the basis for nucleotide selection or catalysis. The way in which the correctly matched and positioned NTP is recognized and how this recognition leads to catalysis remain obscure. The energies of base pairing and stacking are insufficient for base selectivity, and the question arises of why transient occupation of the A site by either incorrect NTP or 2’-dNTP substrates does not lead to erroneous RNA synthesis. Genetic and biochemical studies have implicated two conserved polymerase domains, termed F and G, in the transcription mechanism (Allison et al., 1985
; Archambault et al., 1998
; Bar-Nahum et al., 2005
; Hekmatpanah and Young, 1991
; Thuillier et al., 1996
; Weilbaecher et al., 1994
). Structural studies have identified these two domains with elements adjacent to the polymerase active site, termed the bridge helix (F) and trigger loop (G) (Cramer et al., 2001
). In the X-ray structures of transcribing complexes, however, no contact of these structural elements with NTP in the A or E sites has been observed. Here we report a series of pol II transcribing complex structures that reveal such contacts and suggest the roles of these domains in the transcription mechanism. These structures also address matters arising from the literature concerning the multiplicity of NTP binding sites and the locations of active center magnesium ions.