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

Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule

The Shine-Dalgarno hybrid during initiation of translation and elongation

It was unknown whether a synthetic Shine-Dalgarno (SD) oligonucleotide labelled with ³²P at its 5'-end ([³²P]oct) would be able to reach the anti-SD sequence of 16S rRNA at the early stages of translation only or during elongation. To verify this, [³²P]oct was incubated with 30S ribosomal subunits (RSUs), 70S ribosomes and polysomes, separately, while the SD/anti-SD binding was checked in them through sucrose gradients. The anti-SD sequence resulted highly available in 30S RSUs and sufficiently available in ribosomes. In both 30S RSUs and ribosomes, the addition of a model 002 mRNA in equimolar proportions displaced [³²P]oct for about 50 %. However, in ribosomes the presence of initiation factors (IFs) and fMet-tRNA influence neither the binding of [³²P]oct nor the competition coming from mRNA. In polysomes, [³²P]oct was unable to hybridize the anti-SD sequence, in agreement with the hypothesis that mRNA and 16S rRNA are involved in the SD/anti-SD interaction also during elongation.Невідомо, чи будуть синтетичні олігонуклеотиди Шайна-Дальгарно (SD) мічені ³² P по 5'-кінця ([ ³² P]-окт) досягати анти-SD послідовності 16S рРНК на ранній стадії трансляції або тільки під час елонгації. Дла перевірки даної гіпотези, [ ³² P] окт інкубували з 30S субодиницями рибосом (RSUs) і 70S рибосом і полісом, окремо, SD / анти-SD зв'язування детектувати в градієнти сахарози. Анти-SD послідовності призвело високої доступності в 30-і RSUs і досить доступні в рибосоми. В обох 30S RSUs і рибосом, додавання модель 002 мРНК в еквімолярних кількостей зміщені [ ³² P] окт близько 50%. Тим не менш, в рибосомах присутність факторів ініціації (МФ) і fMet-тРНК не впливають на зв'язування [ ³² P] окт і не конкурують з мРНК. У полісоми, [ ³² P] окт не зміг провести гібридизацію анти-SD послідовності, відповідно до гіпотезою, що мРНК і 16S рРНК беруть участь в SD / анти-SD взаємодія також під час елонгації.Неизвестно, будут ли синтетические олигонуклеотиды Шайна-Дальгарно (SD) меченные ³²P по 5'-концу ([³²P]-окт) достигать анти-SD последовательности 16S рРНК на ранней стадии трансляции или только во время элонгации. Дла проверки данной гипотезы, [³²P] окт инкубировали с 30S субъединицами рибосом (RSUs) и 70S рибосом и полисом, по отдельности, SD / анти-SD связывание детектировали в градиенты сахарозы. Анти-SD последовательности привело высокой доступности в 30-е RSUs и достаточно доступны в рибосомы. В обоих 30S RSUs и рибосом, добавление модель 002 мРНК в эквимолярных количеств смещены [³²P] окт около 50%. Тем не менее, в рибосомах присутствие факторов инициации (МФ) и fMet-тРНК не влияют на связывание [³²P] окт и не конкурируют с мРНК. В полисоме, [³²P] окт не смог провести гибридизацию анти-SD последовательности, в соответствии с гипотезой, что мРНК и 16S рРНК участвуют в SD / анти-SD взаимодействие также во время элонгации

Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion

Site-directed mutagenesis and NMR spectroscopic approaches to the elucidation of the structure-function relationships in translation initiation factors IF1 and IF3.

The recent developments in the knowledge of the structure and structure-function relationships of prokaryotic initiation factors IF1 and IF3 obtained in our laboratory by site-directed mutagenesis, biochemical and NMR-spectroscopic approaches are discussed

Characterization of the Self-Resistance Mechanism to Dityromycin in the Streptomyces Producer Strain

Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain.IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers' microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic's target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli, thereby eliciting antibiotic resistance in this human commensal bacterium

Draft Genome Sequence of Streptomyces sp. Strain AM-2504, Identified by 16S rRNA Comparative Analysis as a Streptomyces kasugaensis Strain

We report here the draft genome sequence of Streptomyces sp. strain AM-2504, a microorganism producing a broad range of biotechnologically relevant molecules. The comparative analysis of its 16S rRNA sequence allowed the assignment of this strain to the Streptomyces kasugaensis species, thus fostering functional characterization of the secondary metabolites produced by this microorganism

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

Structural–Functional Relationship of the Ribonucleolytic Activity of aIF5A from Sulfolobus solfataricus

The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo