PDBsum entry 3d3c

Transcription

PDB id

3d3c

Contents

			Protein chains

					138 a.a.


					85 a.a.

			Waters ×197

References listed in PDB file

Key reference

Title

Structural and functional analysis of the e. Coli nusb-S10 transcription antitermination complex.

Authors

X.Luo, H.H.Hsiao, M.Bubunenko, G.Weber, D.L.Court, M.E.Gottesman, H.Urlaub, M.C.Wahl.

Ref.

Mol Cell, 2008, 32, 791-802. [DOI no: 10.1016/j.molcel.2008.10.028]

PubMed id

19111659

Abstract

Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive antitermination.



	Figure 4. Figure 4. BoxA RNA Binding (A) Mapping of crosslinked peptides on the surface of the NusB-S10^Δloop complex. The view is from the top of Figure 2A. NusB, dark gray; S10, light gray. Crosslinked peptides of NusB (B1, B2, B3; see Table S3 for peptide sequences) are dark blue, cyan, and steel blue, respectively. Crosslinked peptides of S10 (E1 and E3) are red and violet, respectively. Asp118, gold. RNAs encompassing the rrn and λ BoxA elements and used for crosslinking are given above and below the structure, respectively. Boxed regions with residue numbers indicate the core BoxA elements. Residues in green of rrn BoxA RNA and λ BoxA RNA have previously been implicated in recruitment of NusB and S10 to antitermination complexes by mutational analysis (Mogridge et al., 1998). Outlined residues differ in λ BoxA compared to rrn BoxA. Black bars designate crosslinked regions of the RNAs. They are connected by lines to the peptides, to which they have been crosslinked (Table S3). Inset 1 illustrates the deduced topology of the NusB-S10-BoxA RNA complexes. (B) (Top) Representative crosslinking of λ NutR BoxA RNA (left two panels) or rrn BoxA RNA (right two panels) to NusB-S10^Δloop or NusB101-S10^Δloop (NusB^Asp118Asn-S10^Δloop). Two concentrations of protein complex (0.31 and 0.62 μM) were crosslinked, resolved on SDS gels, and visualized by autoradiography. In each panel, RNA alone is in the left lane, NusB-S10^Δloop complex in the central lane, and NusB101-S10^Δloop complex in the right lane. (Bottom) Quantification of crosslink yields. Values are the crosslink yields of the protein components of the NusB101-S10^Δloop samples, relative to the crosslink yields of the corresponding components of the NusB-S10^Δloop samples. The crosslink yields of the components of the NusB-S10^Δloop samples were set at 100% (dashed lines). Values represent the mean of three independent experiments ± the standard errors of the mean. ^p ≤ 0.032; ^*p ≤ 0.020.		Figure 5. Figure 5. NusB-S10^Δloop,Ala86Asp Complex (A) Comparison of the NusB-S10^Δloop complex (left) with the NusB-S10^Δloop,Ala86Asp complex (right). Gray meshes, final 2F[o] − F[c] electron densities covering residue 86 and neighboring residues of the S10^Δloop molecules, contoured at the 1σ level. (Insets) Closeup views of the residue 86 regions. The orientation relative to Figure 2A is indicated. (B) Comparison of the electrostatic surface potentials of the complexes. Blue, positive charge; red, negative charge. Left, NusB-S10^Δloop complex. Right, NusB-S10^Δloop,Ala86Asp complex. The positions of residue 86 are circled. The orientations are the same as in (A).

PROCHECK

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