Elongation factor G (EF‐G) catalyzes the translocation step of protein synthesis in bacteria, and like the other bacterial elongation factor, EF‐Tu‐‐whose structure is already known‐‐it is a member of the GTPase superfamily. We have determined the crystal structure of EF‐G‐‐GDP from Thermus thermophilus. It is an elongated molecule whose large, N‐terminal domain resembles the G domain of EF‐Tu, except for a 90 residue insert, which covers a surface that is involved in nucleotide exchange in EF‐Tu and other G proteins. The tertiary structures of the second domains of EF‐G and EF‐Tu are nearly identical, but the relative placement of the first two domains in EF‐G‐‐GDP resembles that seen in EF‐Tu‐‐GTP, not EF‐Tu‐‐GDP. The remaining three domains of EF‐G look like RNA binding domains, and have no counterparts in EF‐Tu.

Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

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Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 Å: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl-and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA Pro with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA Pro and tRNA Cys with their cognate amino acids.

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