Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

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 antiShine-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.GTPase ͉ KH domain ͉ 30S ribosomal subunit T he bacterial ribosome (70S) is composed of a large (50S) and a small (30S) subunit. The 30S ribosomal subunit (r-subunit) contains an approximately 1,540-nucleotide (nt) rRNA (16S rRNA) and 21 ribosomal proteins (r-proteins). The assembly of each r-subunit involves processing and maturation of rRNA, ordered binding of metal ions and r-proteins, and sequential conformational changes of the complex. Many proteins are associated with this process, including GTPases that represent the largest class of essential ribosome assembly factors in bacteria (1).ERA is the first Ras-like small GTPase found in bacteria (2) and is essential for viability (3). ERA contains an N-terminal GTPase domain and a C-terminal KH domain. The structure of ligand-free Escherichia coli (Ec) ERA (apo-ERA) reveals that the GTPase domain resembles p21 Ras and the KH domain has a type-II fold (4). The structure of Thermus thermophilus (Tt) ERA in complex with the GTP analog GDPNP (ERA-GNP, PDB entry 1WF3) shows that the fold of TtERA is virtually identical to that of EcERA. The GTPase and KH domains are both essential for ERA function. However, the degree of interplay between the two domains, if any, remains unknown (5). ERA binds to 16S rRNA (6, 7) and the 30S r-subunit (8), and is believed to interact with a sequence near the 3Ј end of 16S rRNA (9, 10).The 3Ј end of 16S rRNA plays important roles in the initiation of protein synthesis. For most mRNAs, selection of the correct start codon and translational reading frame is dependent on base pairing between the Shine-Dalgarno (SD) sequence (GGAGG) upstream from the initiator codon in the mRNA and the anti-SD sequence ( 1535 CCUCC 1539 ) nea...

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

“…We used the structural information on GIMAP2 to reanalyze higher-order evolutionary relationships of GTPases and how versions that operate on the membrane might have emerged from an ancestral TRAFAC class GTPase involved in translationrelated functions. Previous sequence-structure analysis suggested that the septins, Tocs, and GIMAPs are further related to GTPases of the Era family, which bind single-stranded 16S rRNA via their C-terminal KH domains and mediate the assembly of the 30S ribosomal subunit (46). Structures of GIMAP2, along with those of the Tocs, strongly support this relationship despite relatively low sequence similarity.…”

Ribosome-associated GTPases: The role of RNA for GTPase activation

“…18 Indeed, it was shown that both isolated 16S rRNA or a 12-nt long mimic of the 16S rRNA 3' end were sufficient to fulfill a GAP-like role on Era. 11,12 However, in contrast to classical GAPs, which insert an Arg into the active site of the GTPase, the Era-bound RNA is not positioned within the catalytic center. This indicates that the RNA induces a structural change on Era that is transmitted to the active site thereby repositioning amino acids to their catalytically competent conformation(s).…”

“…18 In 30S subunit assembly it prevents premature subunit joining and is directly involved in maturation of the 16S ribosomal RNA (rRNA) from the precursor 17S rRNA. 12,[19][20][21] Era has a two-domain arrangement. 22 With its C-terminal KH-domain Era specifically binds 16S rRNA, 11 particularly a short region close to the 3' end of the 16S rRNA, harboring the anti-Shine-Dalgarno sequence and a AUCA motif, which is highly conserved in all three domains of life.…”

“…1). [10][11][12][13][14][15] All of these G proteins have two characteristics in common: First, they all belong to a core of 11 universally conserved GTPases among bacteria, with one exception-EF4 (LepA) is absent in one of 191 bacterial genomes sequenced. 16,17 And second, they are all somehow associated with the ribosome.…”

“…12 The N-terminal domain constitutes the G domain. Era and all other RA GTPases have a hydrophobic amino acid at the position of the catalytic residue in switch II.…”