Joseph Reinbolt

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

Studies of the binding of E. coli ribosomal protein S4 to 16S rRNA after UV irradiation of the S4—16S rRNA complex

In the E. Coli ribosome several proteins directly bind to the 16s rRNA [l-S] . Conversion of these specific non covalent interactions to covalent crosslinks could be of great help for studying the binding sites between a particular protein and the 16s rRNA. It is now well known that upon U.V. irradiation covalent bonds are formed between polynucleotides and proteins. This technique was used to link covalently together DNA and DNA polymerase [6] , synthetic d(A T), and RNA polymerase [7] , aminoacyltRNA synthetase and tRNA [8,9], lac operon and lac repressor [lo] , cyclic AMP and high affinity macromolecular receptors [ 1 l] , polynucleotide and protein in virions [12,13]. We began this study with ribosomal protein S4 and 16s rRNA for different and obvious reasons. The primary structure of 16s rRNA is almost completely determined [ 141. Protein S4 plays an essential role in the Nomura’s assembly map [IS] and its primary sequence is known [ 161; it has been shown that in the specific 16s rRNA-S4 complex, S4 protects as much as one third of the RNA molecule against mild ribonuclease T1 digestion [ 17-201 and this enhances the possibility of covalent cross-links between RNA and protein after U.V. irradiation; the contacts between protein S4 and 16s rRNA have been studied [5,17-221. Recently the lysine residues of protein S4 involved in the binding with 16s rRNA were identified [23] and the C-terminus part of protein S4 was found to be involved in this binding site [24] . The results reported here show the specificity of this cross-linked 16s rRNA-S4 complex after U.V. irradiation; the tryptic peptides of S4 corresponding

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

The primary structure of ribosomal protein S7 from E. coli strains K and B.

Protein S7 from the small subunit of ~sc~ler~chi~ coli ribosomes has been reported to interact specifically with 16 S RNA (reviewed [ 11). The proportion of the RNA involved in the binding has been identified as part of the 3’-end [2-4]. On the other hand the part of the protein that binds to the RNA has been investigated by covalcntly linking S7 to the RNA. Protein 57 can be crosslinked to 16 S RNA by ultraviolet irradiation of the small subunit [5]. Ultraviolet irradiation experiments have also been made on an in vitro complex between protein S7 and 16 S RNA. The results indicated that at least four peptides were crosslinked to the RNA [6]. It can be expected that knowledge of the primary structure of protein S7 will contribute to a deeper understanding of the molecular mechanisms of protein-RNA interaction. It has been shown that E. coli strains differ in their S7 proteins [7-l 11. Ribosomes from strains B, C and MRE600 contain a protein S7 which differs extensively in size, charge, amino acid composition and im~~~unological properties from the protein S7 in strain K [ Ill. The amino acid sequences of protein S7 isolated from E. coli strain B and K are reported here. Protein S7B consists of 153 amino acids and protein S7K contains 177 amino acids. This difference of 24 amino acids between S7B and S7K occurs at the C-terminal end of the protein chain.

Studies of the binding sites of Escherichia coli ribosomal protein S7 with 16S RNA by ultraviolet irradiation

It is now generally agreed that several ribosomal proteins, namely

Crosslinking of elongation factor Tu to tRNAPhe by trans-diamminedichloroplatinum (II) Characterization of two crosslinking sites on EF-Tu

4,

Immunochemical studies of tobacco mosaic virus--V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains.

7,

Localization of two antigenic determinants in histone H4

8, S15,

Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae

20, interact directly with the 16S RNA ofE. coli [1 -5] . The studies of the regions of the specific reconstituted protein-RNA complexes protected against mild enzyme digestion, give some informations about the location of some of these proteins on the 16S RNA molecule [6-11 ]. Further progresses to determine the binding sites between RNA and proteins in the ribosome could be obtained by converting the non-covalent interactions into covalent bonds. Recently photochemical crosslinks between proteins and nucleic acids were obtained in the case of ribosomes. Gorelic was able to link covalently to the RNA practically all the proteins by increasing doses of ultraviolet irradiation in the case of the 30S [12] and the 50S subunits ofE. coli [13]. In a previous paper, we determined the tryptic peptides of protein

Ligand-induced Structural Alterations in Human Iron Regulatory Protein-1 Revealed by Protein Footprinting

4 linked covalently to the 16S RNA in the ultraviolet irradiated 16S RNA-S4 complex [14]. Ribosomal protein