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

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

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

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

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of the cytoplasmic translation initiation factor eIF5b/Fun12. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associate with Fun12 and form 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP-hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation