The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3′ end of 16S rRNA

Abstract

Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides ( 1531 AUCACCUCC 1539 ) near the 3′ end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530 GAUCA 1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530 GA 1531 to 1530 AG 1531 double mutation severely affects the viability of bacteria. However, whether Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.ribosome biogenesis | cell growth regulation

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

“…The N-terminal catalytic domain shares sequence and structural similarity to the adenine DNA methyltransferase (20). Furthermore, two structures of the Aquifex aeolicus KsgA in complex with an RNA fragments of helix 45 were solved (24,25). These structures show catalytically inactive complexes and are inconsistent with structural model based on footprinting data (19).…”

“…7). In the crystal structure of the ternary complex of KsgA and Era bound to a helix 45-containing RNA fragment, the modified adenosines are bound by the C-terminal domain and not the catalytic N-terminal domain (25). These structures do not correspond to catalytically active complexes and might be an artifact of unspecific RNA binding by KsgA.…”

Identification of YbeY-Protein Interactions Involved in 16S rRNA Maturation and Stress Regulation in Escherichia coli

“…Considering a 30S preribosomal intermediate as the substrate, the GTPase Era and nearby ribosomal proteins are plausible candidates for guiding YbeY’s activity (12–14). Tu et al showed how Era can bind to pre-16S rRNA, locking the pre-30S subunit in a conformation that is not favorable for association with the 50S ribosomal subunit and that may facilitate the final processing of 16S rRNA by RNase E, RNase G, and an unknown RNase (29, 46). They proposed that Era acts as a chaperone for processing and maturation of 16S rRNA, as Era releases 30S subunits with mature 16S rRNA after GTP hydrolysis.…”

Crippling the Essential GTPase Der Causes Dependence on Ribosomal Protein L9

“…Der depletion for extended periods causes the accumulation of unstable and/or incomplete large subunits and defects in both 16S and 23S rRNA processing, which others have suggested points to a role in ribosome biogenesis (23)(24)(25)(26)51). Cells with deficient rRNA folding factors commonly display cold sensitivities (46,52). We tested for cold sensitivity in our der mutants and observed none (data not shown).…”