Inter-polysomal coupling of termination and initiation during translation in eukaryotic cell-free system.

The recording of the luciferase-generated luminescence in the eukaryotic cell-free translation system programmed with mRNA encoding firefly luciferase (Luc-mRNA) showed that the addition of free exogenous mRNAs into the translation reactor induces the immediate release of the functionally active protein from the polyribosomes of the translation system. The phenomenon did not depend on the coding specificity of the added free mRNA. At the same time it depended on the "initiation potential" of the added mRNA (including the features that ensure the successful initiation of translation, such as the presence of the cap structure and the sufficient concentration of the added mRNA in the translation mixture). The phenomenon also strictly depended on the presence of the stop codon in the translated mRNA. As the above-mentioned features of the added mRNA imply its activity in initiation of a new translation, the experimental data are found in agreement with the scenario where the molecules of the added mRNA interact by their 5'-ends with terminating and recycling ribosomes, stimulating the release of the complete polypeptides and providing for the initiation of a new translation.

Internal translation initiation and eIF4F/ATP-independent scanning of mRNA by eukaryotic ribosomal particles.

The recombinant mRNAs with 5'-untranslated region, called omega leader, of tobacco mosaic virus RNA are known to be well translated in eukaryotic cell-free systems, even if deprived of cap structure. Using the method of primer extension inhibition (toe-printing), the ribosomal particles were shown to initiate translation at uncapped omega leader when its 5'-end was blocked by a stable RNA-DNA double helix, thus providing evidence for internal initiation. The scanning of the leader sequence and the formation of ribosomal 48S initiation complexes at the initiation AUG codon occurred in the absence of ATP-dependent initiation factor eIF4F, as well as without ATP. The latter results implied the ability of ribosomal initiation complexes for ATP-independent, diffusional wandering (also called bi-directional movement) along the leader sequence during scanning.

Ribosomal protein S1 induces a conformational change of the 30S ribosomal subunit.

A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium bombardment method. Differences in exposure of some ribosomal proteins within the 30S subunit depleted of S1 and within the 30S-S1 complex were found. It was concluded that protein S1 binds in the region of the neck of the 30S ribosomal subunit inducing a conformational change of its structure.

[The androgen receptor: molecular pathology]. / Récepteur des androgènes: pathologie moléculaire.

Androgens play a crucial role in the development, maintenance and regulation of male phenotype and reproductive physiology through the androgen receptor, a transcription factor. Testosterone or dihydrotestosterone binding induces a trans-conformation of the androgen receptor and allows its translocation into the nucleus, where it recognizes specific DNA sequences. Recent developments in molecular genetics, as well as structural analysis of the androgen receptor, allow a better understanding of the structure/function relationship of this nuclear receptor. Molecular analyses of androgen insensitivity syndrome, as well as hormone-resistant prostate cancer, Kennedy's disease and isolated male infertility, have been proved useful as privileged models for this purpose. In the absence of identified AR receptor mutations in androgen insensitivity syndromes, abnormalities of transcriptional cofactor should be considered. Finally, identification of androgen-dependent genes will be helpful for evaluating the degree of the molecular defect of androgen action within target cells.

Mutant luteinizing hormone receptors in a compound heterozygous patient with complete Leydig cell hypoplasia: abnormal processing causes signaling deficiency.

Over the past 5 yr several inactivating mutations in the LH receptor gene have been demonstrated to cause Leydig cell hypoplasia, a rare autosomal recessive form of male pseudohermaphroditism. Here, we report the identification of two new LH receptor mutations in a compound heterozygous case of complete Leydig hypoplasia and determine the cause of the signaling deficiency at a molecular level. On the paternal allele of the patient we identified in codon 343 a T to A transversion that changes a conserved cysteine in the hinge region of the receptor to serine (C343S); on the maternal allele a T to C transition causes another conserved cysteine at codon 543 in trans-membrane segment 5 to be altered to arginine (C543R). Both of these mutant receptors are completely devoid of hormone-induced cAMP reporter gene activation. Using Western blotting of expressed LH receptor protein with a hemagglutinin tag, we further show that despite complete absence of total and cell surface hormone binding, protein levels of both mutant LH receptors are only moderately affected. The expression and study of enhanced green fluorescent protein-tagged receptors confirmed this view and further indicated that initial translocation to the endoplasmic reticulum of these mutant receptors is normal. After that, however, translocation is halted or misrouted, and as a result, neither mutant ever reaches the cell surface, and they cannot bind hormone. This lack of processing is also indicated by reduced presence of an 80-kDa protein, the only N-linked glycosylated protein in the LH receptor protein profile. Thus, complete lack of signaling by the identified mutant LH receptors is caused by insufficient processing from the endoplasmic reticulum to the cell surface and results in complete Leydig cell hypoplasia in this patient.

Molecular action of androgens.

The androgen receptor (AR), which mediates androgen action in the cell, belongs to the superfamily of nuclear receptors, a large group of transcription factors. Recent studies have described how the AR acts on specific target genes. The receptor's specificity of action depends on its regulation at different levels: expression in the cells, ligand binding and DNA-specific sequence recognition by structurally conserved domains and regulation by transcriptional factors in an integrated response. We propose, here, an overview of recent works on the molecular regulation of androgen-dependent genes by AR.