PDBsum entry: 1rio

Transcription/DNA

PDB id: 1rio

Contents

Protein chains

61 a.a. *

97 a.a. *

DNA/RNA

Ligands

MPD

Metals

_CA

Waters ×176

* Residue conservation analysis

PDB id:

1rio

Name:

Transcription/DNA

Title:

Structure of bacteriophage lambda ci-ntd in complex with sig of thermus aquaticus bound to DNA

Structure:

27-mer. Chain: u. Engineered: yes. Other_details: chemically synthesized. 27-mer. Chain: t. Engineered: yes. Other_details: chemically synthesized. Sigma factor siga.

Source:

Synthetic: yes. Thermus aquaticus. Organism_taxid: 271. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Enterobacteria phage lambda. Organism_taxid: 10710. Gene: ci.

Biol. unit:

Pentamer (from PQS)

Resolution:

2.30Å R-factor: 0.218 R-free: 0.254

Authors:

D.Jain,B.E.Nickels,L.Sun,A.Hochschild,S.A.Darst

Key ref:

D.Jain et al. (2004). Structure of a ternary transcription activation complex. Mol Cell, 13, 45-53. PubMed id: 14731393 DOI: 10.1016/S1097-2765(03)00483-0

Date:

17-Nov-03 Release date: 27-Jan-04

Protein chain

Q9EZJ8  (Q9EZJ8_THEAQ) -  RNA polymerase sigma factor

Seq: 438 a.a.

Struc: 61 a.a.

Protein chains

P03034  (RPC1_LAMBD) -  Repressor protein CI

Seq: 237 a.a.

Struc: 97 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Biological process: regulation of transcription, DNA-dependent (2 terms)
• Biochemical function: DNA binding (4 terms)

DOI no: 10.1016/S1097-2765(03)00483-0

Mol Cell 13:45-53 (2004)

PubMed id: 14731393

Structure of a ternary transcription activation complex.

D.Jain, B.E.Nickels, L.Sun, A.Hochschild, S.A.Darst.

ABSTRACT

The cI protein of bacteriophage lambda (lambdacI) activates transcription by binding a DNA operator just upstream of the promoter and interacting with the RNA polymerase sigma subunit domain 4 (sigma(4)). We determined the crystal structure of the lambdacI/sigma(4)/DNA ternary complex at 2.3 A resolution. There are no conformational changes in either protein, which interact through an extremely small interface involving at most 6 amino acid residues. The interactions of the two proteins stabilize the binding of each protein to the DNA. The results provide insight into how activators can operate through a simple cooperative binding mechanism but affect different steps of the transcription initiation process.

Selected figure(s)

Figure 1.
Figure 1. Formation of a Ternary Complex between λcI, Taq σ[4], and DNA(A) Test promoter to detect cooperative binding of λcI and Taq σ[4]. The test promoter is a derivative of placCons-35C (Nickels et al., 2002) that bears a consensus −35 element and modified λO[L]1 operator centered at −45.5 and −55, respectively, upstream of the transcription start site of a modified lac promoter. The −35 element centered at position −45.5 serves as a binding site for the Taq σ[4] moiety (residues 351–438) tethered to the α N-terminal domain and linker (residues 1–248).(B) Effect of λcI on transcription from test promoter in the presence of the α-σ^A chimera. Cells harboring the test promoter and a linked lacZ reporter gene on an F′ episome were transformed with compatible plasmids encoding either λcI (pACλcI4B2) or no λcI (pACΔcI) and either the α-σ^A chimera (pBRα-σ^A) or α (pBRα). Plasmid pBRα-σ^A directs the synthesis of the α-σ^A chimera under the control of an IPTG-inducible promoter, whereas plasmid pACλcI4B2 directs the synthesis of λcI under the control of a constitutive promoter. The cells were grown in the presence of 1 μM IPTG and assayed for β-galactosidase activity.(C) DMS protection assay. A 3′ end-labeled DNA restriction fragment bearing the modified O[L]1 operator and consensus −35 element was incubated with saturating concentrations of λcI alone (lane 3), Taq σ[4] alone (lane 1), λcI and Taq σ[4] (lane 2), or no protein (lane 4) and subjected to DMS treatment followed by piperidine cleavage essentially as described (Sauer et al., 1979). Samples were electrophoresed on a 6% denaturing polyacrylamide gel and the bands visualized by phosphorimaging. The λcI protected guanines at positions 4′, 6′, 7′, and 9′ (consensus [c] half) and enhanced the reactivity of the guanine at position 8′ (nonconsensus [n-c] half), as previously observed (Johnson, 1980). In addition, the guanine at position 3′ in the n-c half (which is not a guanine in the context of wild-type O[L]1) was protected. Taq σ[4] protected a single guanine at position −31′ (bottom strand). Previous DMS protection experiments performed with the σ^70-containing RNAP holoenzyme revealed strong protection of the guanine at promoter position −31′ (Siebenlist et al., 1980).

Figure 3.
Figure 3. Protein/DNA Interactions in the Ternary Complex(A) Schematic representation of protein/DNA interactions, plus λcIB/σ[4] protein/protein interactions, in the ternary complex. The DNA is color coded as in Figure 2A. Colored boxes denote protein residues (dark green, λcIA; light green, λcIB; orange, σ[4]). Connecting black solid lines indicate hydrogen bonds (< 3.2 Å) or salt bridges (< 4 Å) between protein and DNA. The red dashed lines indicate hydrogen bonds and/or salt bridges between λcIB and σ[4]. Thick solid lines indicate more than one hydrogen bond with the same residue. Water molecules are shown as pink spheres. The λcI residues that show symmetric protein/DNA interactions in both monomers of the ternary complex are labeled with an “*.”(B) Comparison of binary and ternary complexes. The ternary complex DNA is shown with the same color coding and in the same orientation as Figure 2A, but as a phosphate backbone worm with base pairs shown as sticks. Proteins are shown as α-carbon backbone worms. The λcI dimer from the ternary complex is colored green, σ[4] orange. The λcI dimer from the λcI/O[L]1 binary complex (Beamer and Pabo, 1992), and the structural core of σ[4] from the σ[4]/−35 element binary complex, each superimposed according to overlapping C1′ atoms of the DNA, are shown in blue and cyan, respectively. Relative movements of the λcI and σ[4] monomers from the binary to ternary complexes are denoted by the thick arrows.(C) The ternary complex DNA is shown with the same color coding as Figure 2A but in a different orientation. The path of the DNA helical axis, calculated using CURVES (Lavery and Sklenar, 1988), is shown for the λcI/σ[4]/DNA ternary complex (green), the λcI/O[L]1 binary complex (blue), and the σ[4]/−35 element binary complex (orange), superimposed on the ternary complex according to overlapping protein α-carbon backbones. The positions of the HTH motifs of λcI (light green) and σ[4] (orange) are shown.

The above figures are reprinted by permission from Cell Press: Mol Cell (2004, 13, 45-53) copyright 2004.

Figures were selected by the author.