Reuben Leberman

A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu

Abstract Polypeptide elongation factor EF-Tu can be isolated from bacterial cell extracts in two fractionation steps. The first is ion-exchange chromatography on DEAE-Sepharose, CL-6B, and the second is gel filtration on AcA 44. The method is illustrated with extracts from Escherichia coli, Bacillus stearothermophilus , and the thermophilic bacterium PS3. The extracts were obtained from lysozyme-treated cells and were processed without high-speed centrifugation or ammonium sulfate fractionation. The procedure is simple and rapid, gives higher yields than previous methods, and is easily scaled to any size preparation. The procedure also produces fractions enriched in the other polypeptide elongation factors EF-Ts and EF-G.

A new additive for protein crystallization

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg‐white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 Å. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 Å. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

1H-NMR SPECTROSCOPY ON ELONGATION-FACTOR TU FROM ESCHERICHIA-COLI - RESOLUTION ENHANCEMENT BY PERDEUTERATION

1H‐NMR Protein EF‐Tu Perdeuteration

Structural requirements of the GDP binding site of elongation factor Tu

Several translation factors of bacterial protein biosynthesis require GTP to perform their function on the ribosome [2]. While much has been found about the specificity of the interaction of translocation factor G [3-51 in respect to its uncoupled GTPase activity and its complex formation with ribosomes, much less information is available about the structural requirements of the GDP binding site of elongation factor Tu. Hamel [6] and Hamel and Cashel [7] found that the natural and synthetic nucleotide analogues pppGpp, dGTP, 3’dGTP and 3’dNH2GTP were similar to GTP in supporting *EF-Tu-dependent N-acetyl-Phe-Phe-tRNA formation, whereas ox GTP, ox-red GTP and ITP were only weakly effective. The corresponding diphosphates ox GDP, ox-red GDP and IDP were found to complex with EF-Tu by the nitrocellulose filter assay somewhat in contrast to the results of Gordon and

31P-NMR spectra of the Ha-ras p21. Nucleotide complexes

Phosphorus nuclear magnetic resonance spectra of the Ha-ras oncogene product p21 and its nucleotide complexes have been obtained. It is shown that the 31P nuclear magnetic resonance spectra of a number of nucleotide-enzyme complexes show some common features. In particular, the chemical shift values of the beta-phosphorus resonance of enzyme-bound NTP and NDP (N = A, G) of hydrolases exhibit a downfield shift virtually identical for myosin, elongation factor Tu, and the Ha-ras oncogene product p21. This suggests that the stereochemistry around the beta-phosphorus might be similar in these compounds.

Archaeabacterial Seryl-tRNA Synthetases: Adaptation to Extreme Environments and Evolutionary Analysis

Abstract. The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA. The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences. In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed. In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred. Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure. The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H. marismortui emerging with the Gram-positive Bacteria, M. jannaschii being near the root of the tree, and S. solfataricus branching with Eucarya.

Crystallization of the seryl-tRNA synthetase-tRNASer complex from Thermus thermophilus

The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A. Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.

Identification of YHR019 in Saccharomyces cerevisiae Chromosome VIII as the Gene for the Cytosolic Asparaginyl-tRNA Synthetase

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl‐tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs−‐1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl‐adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine.