PDBsum entry 3hax

Isomerase/biosynthetic protein/RNA

PDB id: 3hax

Contents

Protein chains

329 a.a. *

53 a.a. *

121 a.a. *

DNA/RNA

Ligands

PGE ×2

EDO ×2

PG4

Metals

_MG ×5

_ZN

Waters ×383

* Residue conservation analysis

PDB id:

3hax

Name:

Isomerase/biosynthetic protein/RNA

Title:

Crystal structure of a substrate-bound gar1-minus h/aca rnp from pyrococcus furiosus

Structure:

Probable tRNA pseudouridine synthase b. Chain: a. Synonym: cbf5, tRNA pseudouridine 55 synthase, psi55 synthase, tRNA- uridine isomerase, tRNA pseudouridylate synthase. Engineered: yes. Ribosome biogenesis protein nop10. Chain: c. Engineered: yes. 50s ribosomal protein l7ae.

Source:

Pyrococcus furiosus. Organism_taxid: 2261. Gene: trub, pf1785. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: pf1141. Gene: rpl7ae, pf1367. Synthetic: yes. Other_details: RNA was prepared by in vitro transcription.

Resolution:

2.11Å R-factor: 0.204 R-free: 0.220

Authors:

K.Ye

Key ref:

J.Duan et al. (2009). Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase. Mol Cell, 34, 427-439. PubMed id: 19481523 DOI: 10.1016/j.molcel.2009.05.005

Date:

03-May-09 Release date: 23-Jun-09

Protein chain

Q7LWY0  (TRUB_PYRFU) -  Probable tRNA pseudouridine synthase B from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 340 a.a.

Struc: 329 a.a.

Protein chain

Q8U1R4  (NOP10_PYRFU) -  Ribosome biogenesis protein Nop10 from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 60 a.a.

Struc: 53 a.a.

Protein chain

Q8U160  (RL7A_PYRFU) -  Large ribosomal subunit protein eL8 from Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)

Seq: 123 a.a.

Struc: 121 a.a.

DNA/RNA chains

G-G-G-U-C-C-G-C-C-U-U-G-A-G-U-G-C-C-C-G-G-G-U-G-A-G-A-G-C-A-U-G-A-U-C-C-C-G-G- 60 bases

A-U-A-A-U-U-FHU-G-A-C-U-C-A-A 14 bases

Enzyme reactions

• Enzyme class: Chain A: E.C.5.4.99.25 - tRNA pseudouridine(55) synthase.
• Reaction: uridine₅₅ in tRNA = pseudouridine₅₅ in tRNA

DOI no: 10.1016/j.molcel.2009.05.005

Mol Cell 34:427-439 (2009)

PubMed id: 19481523

Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase.

J.Duan, L.Li, J.Lu, W.Wang, K.Ye.

ABSTRACT

H/ACA RNAs form ribonucleoprotein complex (RNP) with proteins Cbf5, Nop10, L7Ae, and Gar1 and guide site-specific conversion of uridine into pseudouridine in cellular RNAs. The crystal structures of H/ACA RNP with substrate bound at the active site cleft reveal that the substrate is recruited through sequence-specific pairing with guide RNA and essential protein contacts. Substrate binding leads to a reorganization of a preset pseudouridylation pocket and an adaptive movement of the PUA domain and the lower stem of the H/ACA RNA. Moreover, a thumb loop flips from the Gar1-bound state in the substrate-free RNP structure to tightly associate with the substrate. Mutagenesis and enzyme kinetics analysis suggest a critical role of Gar1 and the thumb in substrate turnover, particularly in product release. Comparison with tRNA Psi55 synthase TruB reveals the structural conservation and adaptation between an RNA-guided and stand-alone pseudouridine synthase and provides insight into the guide-independent activity of Cbf5.

Selected figure(s)

Figure 1.
Figure 1. Overview of Substrate-Bound H/ACA RNP Structures
(A) Secondary structure of H/ACA RNA1 with substrate RNA bound at the pseudouridylation pocket. The lower (P1) and upper (P2) stems, substrate-guide helices PS1 and PS2, and the lower (J1) and upper (J2) three-way junctions are indicated. Base pair symbols are depicted according to a previous rule (Leontis and Westhof, 2001). Hollow letters represent disordered residues in the Gar-minus structure. Prime denotes substrate RNA.
(B) Ribbon representation of the substrate-bound Gar1-minus H/ACA RNP structure, showing Cbf5 PUA domain in light green; Cbf5 catalytic (Cat) domain in dark green; Nop10 in magenta; L7Ae in blue; substrate RNA in purple; guide sequences in orange; the ACA motif, kink-turn motif, and target nucleotide in red; and the remainder of the H/ACA RNA in yellow. The target nucleotide is shown as sticks.
(C) SIGMAA-weighted 2F[o] − F[c] electron density map of the Gar1-minus complex. The 2.1 Å map is contoured at 1 σ. The refined structure is shown as sticks with carbon atoms colored in pink, oxygen in red, nitrogen in blue, phosphorus in orange, and magnesium in green.
(D) The substrate-bound full-complex structure represented as Cα or P traces. The solvent-modified 5 Å electron density map contoured at 1 σ is indicated. Gar1 is colored cyan, and other parts are color coded as in Figure 1B. A symmetry-related molecule, colored in gray, forms a self-duplex with the 5′ extension of H/ACA RNA2.

Figure 2.
Figure 2. Structure and RNP Assembly of Substrate-Bound H/ACA RNA
(A) Structure of substrate-bound H/ACA RNA. Hydrated Mg ions are shown as sticks with Mg colored green and water red.
(B) RNA-binding surface in substrate-bound H/ACA RNP. Shown are the Gar1-minus RNP structure with Gar1 modeled according to its position in the full complex. RNAs are represented as ribbons and planes, and proteins are represented as surfaces. Individual parts are color coded as in Figure 1B. The RNA-binding surface (within 4 Å of RNA) is colored in gray.

The above figures are reprinted by permission from Cell Press: Mol Cell (2009, 34, 427-439) copyright 2009.

Figures were selected by the author.