translational mechanisms and protein synthesis

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Internal translation initiation in the mRNA for the Neurospora crassa albino-3 gene

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA.The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA

Optimization of an in vitro transcription/translation system based on Sulfolobus solfataricus cell lysate.

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pre-transcribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins

IGFBP-3 inhibits Wnt signaling in metastatic melanoma cells.

In previous works, we have shown that insulin-like growth factor-binding protein-3 (IGFBP-3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP-3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti-tumoral effects of IGFBP-3. We show that the anti-tumoral effect of IGFBP-3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP-3 binds the Wnt signalosome interacting specifically with its component GSK-3β. As a consequence, the β-catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK-3β is activated through dephosphorylation, becoming free to target cytoplasmic β-catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP-3 is a novel and effective inhibitor of Wnt signaling. As IGFBP-3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling

Early assembly proteins of the large ribosomal subunit of the thermophilic archaebacterium Sulfolobus. Identification and binding to heterologous rRNA species.

Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent. In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus. The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes. Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified. Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes. By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins

The archaeal elongation factor EF-2 induces the release of aIF6 from 50S ribosomal subunit

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release

The gene of an archaeal α-l-fucosidase is expressed by translational frameshifting

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a α-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a −1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed −1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea