Tài liệu Báo cáo khoa học: Substrate specificity of the pseudouridine synthase RluD in Escherichia

Substrate specificity of the pseudouridine synthase RluD

in Escherichia coli

Margus Leppik, Lauri Peil, Kalle Kipper, Aivar Liiv and Jaanus Remme

Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia

Pseudouridines (Y) are the most common modifications

in stable RNAs. Pseudouridine was discovered as a fifth

nucleotide in yeast tRNA 50 years ago [1]. Pseudo￾uridines are synthesized from uridine by pseudouridine

synthases, a reaction that does not need additional

cofactors or external energy sources. Pseudouridine

synthases are classified into five families according to

their amino acid sequence [2,3]. Despite low sequence

homology of the enzymes, structural comparison of

crystal structures reveals that all pseudouridine synth￾ases share a core with a common fold and a conserved

active site cleft [4].

Pseudouridines are found in all tRNAs and high￾molecular rRNAs. 16S ribosomal RNA from Escheri￾chia coli contains one pseudouridine Y516 formed by

RsuA [5]. 23S rRNA from E. coli contains ten Y resi￾dues, which are made by six enzymes RluA–RluF [6].

Enzymes such as RsuA and RluB isomerize only one

uridine in the substrate RNA whereas others (RluC

and RluD) make three pseudouridines [7–9]. RluA

modifies uridine 746 in 23S rRNA and uridine 32 in

some specific tRNA species [10].

RluD isomerizes uridines at positions 1911, 1915,

and 1917 in stem-loop 69 (H69) of 23S rRNA [8,9].

Y1917 is found at the corresponding position of the

large ribosomal subunit RNAs throughout all king￾doms. It is the most conserved modification in

rRNA [6]. Y1915 is also highly conserved [6]. Y1915

is methylated at N3 in several eubacteria [11]. Y1911

is also well conserved, except in archaea [6]. Y to C

mutation at position 1917 has a dramatic effect on

the ribosome functioning, which is explained by the

universal nature of Y1917 [12]. H69 of 23S rRNA

directly interacts with tRNA at the A and P site

[13,14]. H69 forms the intersubunit bridge 2 with

helix 44 of 16S rRNA [15,16]. Y1917 forms a reverse

Hoogsteen base pair with A1912, which in turn

forms A-minor interaction with base pairs C1407–

G1494 of 16S rRNA [16]. Pseudouridine residues can

stabilize the 3D RNA structure as revealed by

Keywords

23S rRNA; helix 69; pseudouridine;

ribosome assembly; RluD

Correspondence

J. Remme, Riia 23, 51010 Tartu, Estonia

Fax: +372 42 0286

Tel: +372 73 75031

E-mail: [email protected]

(Received 24 May 2007, revised 6 Septem￾ber 2007, accepted 10 September 2007)

doi:10.1111/j.1742-4658.2007.06101.x

Pseudouridine synthase RluD converts uridines at positions 1911, 1915,

and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in

the functionally important helix-loop 69 of 23S rRNA. RluD is the only

pseudouridine synthase that is required for normal growth in Escherichia

coli. We have analyzed substrate specificity of RluD in vivo. Mutational

analyses have revealed: (a) RluD isomerizes uridine in vivo only at posi￾tions 1911, 1915, and 1917, regardless of the presence of uridine at other

positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of

one U by C has no effect on the conversion of others (i.e. formation of

pseudouridines at positions 1911, 1915, and 1917 are independent of each

other); (c) A1916 is the only position in the loop of helix 69, where muta￾tions affect the RluD specific pseudouridine formation. Pseudouridines

were determined in the ribosomal particles from a ribosomal large subunit

defective strain (RNA helicase DeaD–

). An absence of pseudouridines in

the assembly precursor particles suggests that RluD directed isomerization

of uridines occurs as a late step during the assembly of the large ribosomal

subunit.

Abbreviations

Y, pseudouridine; ASL, anticodon stem loop; H69, stem-loop 69.

FEBS Journal 274 (2007) 5759–5766 ª 2007 The Authors Journal compilation ª 2007 FEBS 5759