Biomedical subjects

R Nageotte

Publications and source records attributed to R Nageotte.

6 recordsLinked to original sources

PubMed1996

Destabilizing interactions between the partners of a bifunctional fusion protein.

Hybrid MalE-GVP is a bifunctional protein in vitro since it binds maltose as protein MalE of Escherichia coli and since it is dimeric and specifically binds single-stranded DNA as protein GVP of phage M13. The oxidation rate of a unique cysteine residue was used to compare the stabilities of GVP in its free and hybrid forms, under conditions where MalE was either folded or unfolded by a denaturing agent. The results showed that both the covalent link and tertiary non-covalent interactions between MalE and GVP destabilized GVP in MalE-GVP. To test whether GVP had identical structures in its free and hybrid forms, mutations were used as local conformational probes. The effects of these mutations on the capabilities of MalE-GVP to dimerize and to bind single-stranded DNA were assayed in vitro. They were compatible with the effects of the same mutations on the global activity of free GVP in vivo and with the effects that could be predicted from the known data on free GVP, in particular its crystal structure. Thus, one partner of a hybrid protein can be destabilized by the other partner while maintaining its structural and functional characteristics.

ATP-Binding Cassette Transporters↗

PubMed1995

Macromolecular recognition through electrostatic repulsion.

In the process of genetic translation, each aminoacyl-tRNA synthetase specifically aminoacylates its cognate tRNAs and rejects the 19 other species of tRNAs. A decrease in the specificity of this reaction can result in misincorporations of amino acids into proteins and be deleterious to the cell. In the case of tyrosyl-tRNA synthetase from Bacillus stearothermophilus, the change of residue Glu152 into Ala results in erroneous interactions with non-cognate tRNAs. To analyse how Glu152 contributes to the discrimination between tRNAs by tyrosyl-tRNA synthetase, 11 changes to this residue were created by mutagenesis. The misaminoacylations of tRNA(Phe) and tRNA(Val) with tyrosine in vitro (on a scale going from 1 to 30) and the toxicity of tyrosyl-tRNA synthetase in vivo (on a scale from 1 to 10(7)) increased in a correlated way when the nature of the side chain in position 152 varied from negatively charged to uncharged then to positively charged. The aminoacylation of tRNA(Tyr) was unaffected by the mutations. The results show that the role of Glu152 in the discrimination between tRNAs is purely negative, that it acts by electrostatic repulsion of non-cognate tRNAs and that this mechanism has been conserved throughout evolution.

Biological Evolution↗

PubMed1993

Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase.

We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNA(Tyr) by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling. We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS. Comparison of the crystal structures of TyrRS and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNA(Tyr) and tRNA(Gln) indicate that the two synthetases bind their cognate tRNAs differently. The mutagenesis data on tRNA(Tyr) confirm the model of the TyrRS:tRNA(Tyr) complex on the following points. TyrRS approaches tRNA(Tyr) on the side of the variable loop. The bases of the first three pairs of the acceptor stem are not recognized. The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer. The anticodon is the major identity element of tRNA(Tyr). We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs. Using this approach, we have shown that residue Glu152 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions. The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs. The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for folding and function, in particular for the specific recognition and charging of tRNA(Tyr).

Binding Sites↗

PubMed1981

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs.

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.

Animals↗

PubMed1981

Molecular cloning of a mouse submaxillary gland renin cDNA fragment.

The mRNA encoding mouse renin has been partially purified from total poly(A)-containing RNA of submaxillary glands of male Swiss mice. Corresponding cDNAs were cloned in the Pst I site of pBR322. Recombinants have been characterized by differential screening and hybrid-arrested translation. The DNA of clone pRn3-5 has been used to study the expression of renin mRNA in the submaxillary gland and in the kidney of different mouse strains. The renin mRNA from submaxillary gland and kidney have the same length (1600 nucleotides) and appear to be the products of the same gene. In vitro translation of mRNAs and RNA blotting experiments have shown that renin mRNA sequences are accumulated in the submaxillary gland of males of AKR and Swiss strains but not in the gland of male BALB/c.

Animals↗

PubMed1980

Mouse immunoglobulin genes: a bacterial plasmid containing the entire coding sequence for a pre-gamma 2a heavy chain.

A DNA sequence complementary to the entire coding part of a mouse gamma 2a immunoglobulin heavy chain mRNA isolated from a myeloma producing a levan binding protein (UPC 10), has been cloned in the PstI site of pBR 322. Transformants containing sequences complementary to purified gamma 2a heavy chain mRNA were selected. One transformant, pG2a-10-21, containing a 1750 nucleotide insert, has been characterized by hybrid-arrested translation and purification of gamma 2a heavy chain mRNA on DNA-DBM cellulose filters. Restriction enzyme analysis and partial sequencing demonstrate that the pG2a-10-21 contains the complete structural sequence for the gamma 2a heavy chain and predicts the sequence of a 18 amino acid hydrophobic amino terminal extra piece segment.

Animals↗