PDBsum entry 1l9z

Transcription/DNA

PDB id: 1l9z

Contents

Protein chains

224 a.a.

1084 a.a.

1183 a.a.

92 a.a.

319 a.a.

DNA/RNA

Metals

_ZN ×2

_MG

References listed in PDB file

Key reference

Title

Structural basis of transcription initiation: an RNA polymerase holoenzyme-Dna complex.

Authors

K.S.Murakami, S.Masuda, E.A.Campbell, O.Muzzin, S.A.Darst.

Ref.

Science, 2002, 296, 1285-1290. [DOI no: 10.1126/science.1069595]

PubMed id

12016307

Abstract

The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (alpha2betabeta'omegasigmaA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the sigma subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the sigma subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

Figure 1.
Fig. 1. Fork-junction DNA and electron density map. (A) Synthetic DNA oligonucleotides used for complex formation and crystallization. The numbers above denote the DNA position with respect to the transcription start site at +1. Downstream corresponds to the direction of RNAP movement during transcription. Mutations in the bottom DNA strand cause corresponding mutations in the RNA transcript, defining it as the template (versus the nontemplate) strand. The DNA sequence is derived from the full con promoter (4), with -35 and -10 elements (shaded yellow and labeled) as well as an extended -10 element (shaded red and labeled). (B) Stereo view of the Taq RNAP holoenzyme/fork-junction DNA complex. The -carbon backbone of is colored white, cyan, ' pink, and orange (the subunits are not visible). The DNA template strand is colored dark green, and the nontemplate strand is light green, except for the -35 and -10 elements, which are colored yellow. The visible structural domains of ( [2] and [4]) (1, 9) are labeled. The direction of transcription (downstream) is to the right. The experimental electron density map, calculated using observed amplitude (F[o]) coefficients, is shown (blue net, contoured at 1.5 ), and was computed using multiple isomorphous replacement phases (Table 1), followed by density modification. The view is sliced at a level just in front of the DNA to reveal the ' NH[2]-terminal Zn2+-binding domain and the associated Zn2+ (labeled, shown as a green sphere). Shown in red is a difference Fourier map, calculated using (|F[o]EMTS - F[o]native|) coefficients (Table 1), revealing the Hg-binding site that was used to locate the Zn2+-site.

Figure 3.
Fig. 3. Conformational changes. The superimposed -carbon backbones of the Taq RNAP holoenzyme alone (1) and the holoenzyme within the fork-junction DNA complex are shown as worms (view the same as Fig. 2A). The structure of holoenzyme alone is colored gray (core RNAP) and black ( ). The two modules that move in the holoenzyme-DNA complex as compared with the holoenzyme alone are colored as follows: clamp + [2], magenta and orange (respectively); flap + [4], blue and orange (respectively). The phosphate backbones of the DNA in the holoenzyme/DNA complex are shown as ribbons and colored green (template strand, t) and light green (nontemplate strand, nt). The downstream direction is indicated. The movements of the mobile modules from the holoenzyme structure to their positions in the holoenzyme-DNA complex are indicated by the arrows.

The above figures are reprinted by permission from the AAAs: Science (2002, 296, 1285-1290) copyright 2002.