Discrete determinants in transfer RNA for editing and aminoacylation.

During translation errors of aminoacylation are corrected in editing reactions which ensure that an amino acid is stably attached to its corresponding transfer RNA (tRNA). Previous studies have not shown whether the tRNA nucleotides needed for effecting translational editing are the same as or distinct from those required for aminoacylation, but several considerations have suggested that they are the same. Here, designed tRNAs that are highly active for aminoacylation but are not active in translational editing are presented. The editing reaction can be controlled by manipulation of nucleotides at the corner of the L-shaped tRNA. In contrast, these manipulations do not affect aminoacylation. These results demonstrate the segregation of nucleotide determinants for the editing and aminoacylation functions of tRNA.

Protein synthesis editing by a DNA aptamer.

Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response.

Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step.

The hydrolysis of single-stranded DNA catalyzed by wild-type staphylococcal nuclease (SNase) and two mutants has been studied as a function of both pH and solvent viscosity. The kcat for wild-type SNase increases with pH; the slope of the plot of log kcat vs pH = 0.45 +/- 0.01. The dependence of kcat/Km on pH for wild-type SNase is biphasic with a break at pH approximately 8: for pH < or = 8, the plot of log kcat vs pH is linear with a slope = 1.20 +/- 0.06; for pH > or = 8, the slope = 0.00 +/- 0.04. The dependencies of both kcat and kcat/Km on solvent viscosity are also pH-dependent: below pH 7.3, both kinetic parameters are independent of solvent viscosity; above pH 7.3, both are inversely proportional to solvent viscosity. Thus, at pH 9.5, where SNase is routinely assayed, the rate-determining steps for both kcat and kcat/Km are external steps (product dissociation for kcat and substrate binding for kcat/Km) and not an internal step (e.g., hydrolysis of the phosphodiester bond). We have also studied the E43D mutant in which the putative active-site general basic catalyst Glu-43 is replaced with Asp. From pH 7.5 to pH 9.5, both log kcat and log (kcat/Km) are directly proportional to pH (slopes = 1.01 +/- 0.03 and 0.95 +/- 0.04, respectively) and independent of solvent viscosity. At pH 9.5, the rate-determining step is an internal step.(ABSTRACT TRUNCATED AT 250 WORDS)

1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain.

The structure of the B2 immunoglobulin-binding domain of streptococcal protein G has been determined at 1.67-A resolution using a combination of single isomorphous replacement (SIR) phasing and manual fitting of the coordinates of the NMR structure of B1 domain of streptococcal protein G [Gronenborn, A. M., et al. (1991) Science 253, 657-661]. The final R value was 0.191 for data between 8.0 and 1.67 A. The structure described here has 13 residues preceding the 57-residue Ig-binding domain and 13 additional residues following it, for a total of 83 residues. The 57-residue binding domain is well-determined in the structure, having an average B factor of 18.0. Only residues 8-77 could be located in the electron density maps, with the ends of the structure fading into disorder. Like the B1 domain, the B2 domain consists of four beta-strands and a single helix lying diagonally across the beta-sheet, with a -1, +3 chi, -1 topology. This small structure is extensively hydrogen-bonded and has a relatively large hydrophobic core. These structural observations may account for the exceptional stability of protein G. A comparison of the B2 domain X-ray structure and the B1 domain NMR structure showed minor differences in the turn between strands and two and a slight displacement of the helix relative to the sheet. Hydrogen bonds between crystallographically related molecules account for most of these differences.

Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability.

The high-resolution X-ray structure of wild-type staphylococcal nuclease (E43 SNase) suggests that Glu 43 acts a general basic catalyst to assist the attack of water on a phosphodiester substrate [Loll, P., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183]. Glu 43 is located at the base of the solvent-exposed and conformationally mobile omega-loop in the active site of E43 SNase having the sequence Glu43-Thr44-Lys45-His46-Pro47-Lys48- Lys49-Gly50-Val51-Glu52, where the gamma-carboxylate of Glu 52 is hydrogen bonded to the amide hydrogen of Glu 43. With a metabolic selection for SNase activity produced in an Escherichia coli host, we detected an unexpected deletion of residues 44-49 of the omega-loop of E43 SNase in cassette mutagenesis experiments designed to randomize codons 44 and 45 in the omega-loop and increase the activity of the previously described E43D mutation (D43 SNase). A high-resolution X-ray structure of D43 SNase has revealed that the E43D substitution significantly changes the structure of the omega-loop, reduces the interaction of the essential Ca2+ ion with its active-site ligands, and diminishes the network of hydrogen-bonded water molecules in the active site [Loll, P., & Lattman, E. E. (1990) Biochemistry 29, 6866]. This deletion of six amino acids from the omega-loop generates a protein (E43 delta SNase) having a partially solvent-exposed, surface beta-turn with the sequence Glu43-Gly50-Val51-Glu52; the structure of this beta-turn is addressed in the following article [Baldisseri et al. (1991) Biochemistry (following paper in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)