Structure of ERA in complex with the 3' end of 16S rRNA: implications for ribosome biogenesis - PubMed (original) (raw)
Structure of ERA in complex with the 3' end of 16S rRNA: implications for ribosome biogenesis
Chao Tu et al. Proc Natl Acad Sci U S A. 2009.
Abstract
ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a "twist" for noneukaryotic ERA proteins by also recognizing the CCUCC.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Structure-based sequence alignment of AaERA, TtERA and EcERA (GenBank accession codes NP214369, YP143386 and YP853700, respectively). Secondary structural elements and the position of the interdomain 17-aa linker are indicated above the sequences. The G1-G5 regions of the GTPase domain are indicated below the sequences. Switch regions I and II and the GXXG and variable loops are indicated. Identical residues and similar residues are shaded in dark and light green, respectively. Identical residues in any two of the three sequences are shaded in light blue. Residues in the ERA-GNP-RNA structure which interact with RNA are indicated with arrows. Detailed protein-RNA interactions are illustrated in
Fig. S3
.
Fig. 2.
Structure and conformation of ERA. (A) Four crystal structures are shown based on superimposed GTPase domains, including ERA-GNP-RNA (this work) and ERA-GNP (PDB entry 1WF3) sharing conformation 1, and apo-ERA (PDB entry 1EGA) and ERA-GDP (this work) sharing conformation 2. The color scheme is the same as in
Fig. S1
, except that the βa and β7 strands are also highlighted in red. (B) Stereoview of superimposed ERA-GNP-RNA (in orange) and ERA-GDP (in cyan), showing dramatic differences between conformations 1 and 2.
Fig. 3.
ERA-GNP interactions in the ERA-GNP-RNA structure. (A) Stereoview showing the details of protein-GNP interactions. GNP is shown as a stick model in atomic colors (carbon, green; oxygen, red; nitrogen, blue; and phosphorus, orange). Amino acid residues are shown as stick models in the same atomic colors except that carbon is in gray. H-bonds are indicated with dashed lines. Three water molecules are shown and labeled as Wat1, Wat2, and Wat3, respectively. (B) Stereoview showing the details around Wat3. Schematic representation and color scheme are the same as in A. Distances less than or equal to 3.5 Å from Wat3 to neighboring atoms are indicated with thin, solid lines.
Fig. 4.
The ERA KH domain with or without RNA. (A) Stereoview showing the KH superposition between ERA-GNP-RNA (in orange) and ERA-GDP (in cyan). RNA is shown as sticks in atomic colors (carbon, orange; nitrogen, blue; oxygen, red; and phosphorus, dark orange). (B) Electrostatic-surface representation of the KH domain and the RNA in ERA-GNP-RNA. (C) Surface representation of the KH domain in ERA-GDP. In B and C, the orientation of the complex is related to the orientation in A by an approximate 90° rotation around the vertical axis; positively charged areas are indicated in blue and negatively charged areas in red.
Fig. 5.
The KH-RNA interactions. Stereoview showing the KH-RNA complex in the ERA-GNP-RNA structure. Seven anchor-point side chains are shown as sphere models. Residues involved in RNA recognition are colored in yellow. RNA is shown as a stick model in atomic colors (carbon, gray; oxygen, red; and nitrogen, blue) except that U1540 and U1541 are shown in gray.
Fig. 6.
Schematic illustration for the functional cycle of ERA. The GTPase domain is represented by a gray rectangle, the KH domain by an orange oval, GTP and GDP by purple cartoons, and the conformations of ERA by numbers in red. The pre-30S particle and 30S r-subunit are represented by larger gray ovals. The pre-16S rRNA (an RNase III cleavage product with a 26-bp stem and a 2-nt 3′ overhang as indicated) and 16S rRNA are represented by a gray line with embedded 1531AUCACCUCCUUA1542 sequence at the 3′ end. The unoccupied ERA-binding pocket in the pre-30S particle and that in the 30S r-subunit are indicated in white. The four functional states, including (A) apo-ERA, (B) ERA-GTP, (C) ERA-GTP-pre-30S, and (D) ERA-GDP, are represented by the apo-ERA (PDB entry 1EGA), ERA-GNP (PDB entry 1WF3), ERA-GNP-RNA (this work), and ERA-GDP (this work) structures. In C, the cleavage sites of RNase E, RNase G and the unknown nuclease are indicated with numbered arrows 1, 2 and 3, respectively. (E) The pre-30S particle contains pre-16S rRNA. (F) The mature 30S r-subunit contains 16S rRNA. (G) The mature 30S r-subunit may bind apo-ERA in a distinct conformation 3 (PDB entry 1X1L).
References
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- Hang JQ, Zhao GS. Characterization of the 16S rRNA- and membrane-binding domains of Streptococcus pneumoniae Era GTPase - Structural and functional implications. Eur J Biochem. 2003;270:4164–4172. - PubMed
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