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Review
. 2009 Mar;10(3):817-834.
doi: 10.3390/ijms10030817. Epub 2009 Mar 2.

The role of disordered ribosomal protein extensions in the early steps of eubacterial 50 S ribosomal subunit assembly

Youri Timsit  1 , Zahir Acosta  1 , Frédéric Allemand  2 , Claude Chiaruttini  2 , Mathias Springer  2
Affiliations
Review

The role of disordered ribosomal protein extensions in the early steps of eubacterial 50 S ribosomal subunit assembly

Youri Timsit et al. Int J Mol Sci. 2009 Mar.

Abstract

Although during the past decade research has shown the functional importance of disorder in proteins, many of the structural and dynamics properties of intrinsically unstructured proteins (IUPs) remain to be elucidated. This review is focused on the role of the extensions of the ribosomal proteins in the early steps of the assembly of the eubacterial 50 S subunit. The recent crystallographic structures of the ribosomal particles have revealed the picture of a complex assembly pathway that condenses the rRNA and the ribosomal proteins into active ribosomes. However, little is know about the molecular mechanisms of this process. It is thought that the long basic r-protein extensions that penetrate deeply into the subunit cores play a key role through disorder-order transitions and/or co-folding mechanisms. A current view is that such structural transitions may facilitate the proper rRNA folding. In this paper, the structures of the proteins L3, L4, L13, L20, L22 and L24 that have been experimentally found to be essential for the first steps of ribosome assembly have been compared. On the basis of their structural and dynamics properties, three categories of extensions have been identified. Each of them seems to play a distinct function. Among them, only the coil-helix transition that occurs in a phylogenetically conserved cluster of basic residues of the L20 extension appears to be strictly required for the large subunit assembly in eubacteria. The role of alpha helix-coil transitions in 23 S RNA folding is discussed in the light of the calcium binding protein calmodulin that shares many structural and dynamics properties with L20.

Keywords: Flexibility; calmodulin; electrostatic; helix unwinding; helix-coil; linker; structural transitions; unfolding.

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Figures

Figure 1.
Figure 1.
Stereo view of the large subunit of T. thermophilus [14] showing the distribution of the six ribosomal proteins L3 (purple blue), L4 (orange), L13 (green), L20 (magenta), L22 (cyan) and L24 (red) essential for the formation of the first reconstitution intermediate in vitro.
Figure 2.
Figure 2.
Comparison of the six proteins that are essential for the early steps of the assembly of the 50 S subunit of the eubacterial ribosome. a. L3. b. L4. c. L13. d. L20. e. L22. f. L24. The structures of the proteins bound to the ribosome are depicted in yellow and the free forms, when available, are depicted in purple blue. In the crystal structure of the free L20, two folded states coexist within the unit cell: a partially unfolded form (cyan) and a folded form (purple blue) (see also Figure 3).
Figure 3.
Figure 3.
Crystal structure of unbound L20 protein. Stereoview of the heterotetramer of the asymmetric unit. The two folded monomers are represented by blue and cyan ribbons. The two partly unfolded monomers are represented by green and yellow ribbons. The unfolded regions of the α2 helix are represented in red. The folded and unfolded dimers mutually stabilise each other.
Figure 4.
Figure 4.
(a) The path of L20 within the core of the large subunit of T. thermophilus [14] (as a representative structure for eubacteria). The box represents the region that is zoomed in b. (b) The interaction of the basic residues (blue) of cluster of the helix α2 of L20 with the 23 S RNA within the 50 S subunit of E. coli (up) [15] and of T. thermophilus (bottom) [14]. The numbering of residues is shifted to fit to the sequence of A. aeolicus (-2 aas).
Figure 5.
Figure 5.
(a) Sequence alignment of the ribosomal protein L20 of representative species of eubacteria. The corresponding secondary structure elements are indicated above in cyan for the partially unfolded free crystal form and in orange for the folded ribosomal form. (b) Disorder prediction calculated with the program PondR for three species. Red: A. aeolicus. Blue; E. coli. Green: D. radiodurans. (c) Ribbon representation of the ribosomal form of T. thermophilus. The N-ter extension is represented in blue and the C-ter globular domain is represented in green.
Figure 6.
Figure 6.
Charge repulsion and unwinding of α-helices. Comparison of the folded (left) and the partially unfolded (right) forms of L20 (2GHJ)(a), calmodulin (1CCL and 2IX7) (b) and poplar thioredoxin (2P5Q and 2P5R)(c). The cluster of charged residues are indicated by parentheses. The unfolded regions are indicated with an arrow. In each case, the electrostatic repulsion between the side chains of the charged residues is thought to be responsible for the helical unwinding. The unfolded form of L20 is stabilised through intermolecular contacts with the folded form in the crystal packing (Figure 3).
Figure 7.
Figure 7.
Structural rearrangements found at the interface between the globular domain and the extension of L20 of A. aeolicus. (a) The partially unfolded form is stabilised by the formation of two salt bridges that link two evolutionary conserved basic residues arg 90 and lys 91 to asp 95 and glu 87 located within the globular domain. (b) In the folded form, the two salt bridges are disrupted and the side chains of arg 90 and lys 91 embrace a sulfate ion. The sulfate ion occupies a position identical to a phosphate group of the H40/41 helix within the 50 S subunit of the eubacterial ribosome. In the unfolded form (a), due to the polarity of the helix (α4), the sulfate ion is also maintained at its N-ter extremity.
Figure 8.
Figure 8.
Fishing mechanism that can help to bring into proximity RNA segments coming from different rRNA domains. The two forms of L20 display very different electrostatic surface potential and affinity for RNA (top: partially unfolded, bottom: folded).
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