Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure
Classification:
Lyase/RNA
Technique:
X-Ray Diffraction
Resolution:
2.05
R value free:
0.263
R value observed:
0.234
R value work:
0.234
Abstract of the PDB Structure's related Publication:
RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adopting its canonical U turn conformation, the anticodon loop folds into a new structure with a reverse-Hoogsteen base pair and three flipped-out nucleotides. Sequence conservation, the cocrystal structure, and the results of structure-guided mutagenesis suggest that RluA recognizes its substrates indirectly by probing RNA loops for their ability to adopt the reorganized fold. The planar, cationic side chain of an arginine intercalates between the reverse-Hoogsteen base pair and the bottom pair of the anticodon stem, flipping the nucleotide to be modified into the active site of RluA. Sequence and structural comparisons suggest that pseudouridine synthases of the RluA, RsuA, and TruA families employ an equivalent arginine for base flipping.
RluA forms pseudouridines at positions 746 in the loop of hairpin 35 of 23S ribosomal RNA (Domain II) and 32 in the loop of anticodon branch of a few E. coli tRNA. It reacts efficiently with full-length 23S RNA, a rRNA fragment encompassing the hairpin 35 (residues 1-847) and the mini tRNA comprising the anticodon stem-loop. RluA induces a dramatic reorganization of the RNA substrate allowing the target U to flip into the active site of the enzyme. Pseudouridylation occurs in single-stranded segments closed by a stem. The enzyme has no dependence on Mg2+, the rate of U-isomerisation is even faster in EDTA than in the presence of Mg2+. E. coli RluA belongs to the same subgroup of RNA pseudouridine synthases as E. coli, RluC, RluD and TruC as well as S. cerevisiae Pus5, Pus6, Pus8 and Pus9.
A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).
Wrzesinski J, Nurse K, Bakin A, Lane BG, Ofengand J
The Handling of the Mechanistic Probe 5-Fluorouridine by the Pseudouridine Synthase TruA and Its Consistency with the Handling of the Same Probe by the Pseudouridine Synthases TruB and RluA.
McDonald MK, Miracco EJ, Chen J, Xie Y, Mueller EG
A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).
Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure.
Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA.
Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine.
Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis.
The Handling of the Mechanistic Probe 5-Fluorouridine by the Pseudouridine Synthase TruA and Its Consistency with the Handling of the Same Probe by the Pseudouridine Synthases TruB and RluA.