scholarly journals In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon Sulfolobus solfataricus

2001 ◽  
Vol 183 (13) ◽  
pp. 3866-3874 ◽  
Author(s):  
Andrea Ciammaruconi ◽  
Paola Londei
Keyword(s):  
16S Rrna ◽  
Sulfolobus Solfataricus ◽  
Conserved Sequence ◽  
Processing Enzymes ◽  
Cell Extracts ◽  
In Vitro Processing ◽  
16S Rna ◽  
Thermophilic Archaeon ◽  
Rna Maturation

ABSTRACT In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5′ external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdomCrenarchaeota but apparently not in theEuryarchaeota. Furthermore, S. solfataricuspre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and theEuryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5′ or the 3′ end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.

scholarly journals Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Archaea ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Giada Lo Gullo ◽  
Rosanna Mattossovich ◽  
Giuseppe Perugino ◽  
Anna La Teana ◽  
Paola Londei ◽  
...  
Keyword(s):  
High Efficiency ◽  
Specific Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Essential Element ◽  
23S Rrna ◽  
Translation System ◽  
Cell Lysate ◽  
Cell Extracts

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 pretranscribed 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.

scholarly journals Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

2004 ◽  
Vol 48 (6) ◽  
pp. 1993-1999 ◽  
Author(s):  
Vidula Dixit ◽  
Elisabetta Bini ◽  
Melissa Drozda ◽  
Paul Blum
Keyword(s):  
Transcription Factor ◽  
Mercuric Chloride ◽  
Sulfolobus Solfataricus ◽  
In Vitro Transcription ◽  
Mercury Toxicity ◽  
Transcription Activity ◽  
Treated Cell ◽  
Mercuric Ion ◽  
Cell Extracts

ABSTRACT Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms. Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at submicromolar concentrations and a cytocidal response at micromolar concentrations. The cytostatic response was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several transcripts, including tfb-1, tfb-2, and lacS. Whole-cell extracts prepared from mercuric chloride-treated cells or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters. Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting factors in the mercury-treated cell extracts. Addition of transcription factor B (TFB), a general transcription factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored >50% of in vitro transcription activity. Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore the in vitro transcription activity of TFB-immunodepleted cell extracts. These findings indicate that the toxicity of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation.

scholarly journals Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 3' end formation in vitro.

1987 ◽  
Vol 7 (5) ◽  
pp. 1663-1672 ◽  
Author(s):  
K L Mowry ◽  
J A Steitz
Keyword(s):  
Histone H3 ◽  
Nuclear Extract ◽  
Sequence Element ◽  
Conserved Sequence ◽  
In Vitro Processing ◽  
Sequence Elements ◽  
Rna Fragments ◽  
Sm Protein ◽  
Small Nuclear Ribonucleoproteins

Pre-mRNA substrates containing sequences from human and mouse histone genes are accurately processed in a HeLa cell nuclear extract to generate mature 3' termini. When in vitro processing reactions containing either human histone H3 or mouse histone H3 transcripts are treated with RNase T1 and probed with antibodies specific for the Sm protein determinants or for the trimethylguanosine cap structure unique to the U RNAs present in small nuclear ribonucleoproteins, RNA fragments that encompass the site of 3' end formation on the pre-mRNA transcript are selectively recovered. Several different interactions are detected: at time zero, the protected region contains the upstream conserved hairpin loop structure; at later times during the reaction, protection extends beyond the site of 3' end formation to include the downstream conserved sequence element and the 5' cap of the transcript is bound as well. Possible interactions between Sm small nuclear ribonucleoproteins and these conserved sequence elements in histone pre-mRNAs are discussed.

Both conserved signals on mammalian histone pre-mRNAs associate with small nuclear ribonucleoproteins during 3' end formation in vitro

1987 ◽  
Vol 7 (5) ◽  
pp. 1663-1672
Author(s):  
K L Mowry ◽  
J A Steitz
Keyword(s):  
Histone H3 ◽  
Nuclear Extract ◽  
Sequence Element ◽  
Conserved Sequence ◽  
In Vitro Processing ◽  
Sequence Elements ◽  
Rna Fragments ◽  
Sm Protein ◽  
Small Nuclear Ribonucleoproteins

Pre-mRNA substrates containing sequences from human and mouse histone genes are accurately processed in a HeLa cell nuclear extract to generate mature 3' termini. When in vitro processing reactions containing either human histone H3 or mouse histone H3 transcripts are treated with RNase T1 and probed with antibodies specific for the Sm protein determinants or for the trimethylguanosine cap structure unique to the U RNAs present in small nuclear ribonucleoproteins, RNA fragments that encompass the site of 3' end formation on the pre-mRNA transcript are selectively recovered. Several different interactions are detected: at time zero, the protected region contains the upstream conserved hairpin loop structure; at later times during the reaction, protection extends beyond the site of 3' end formation to include the downstream conserved sequence element and the 5' cap of the transcript is bound as well. Possible interactions between Sm small nuclear ribonucleoproteins and these conserved sequence elements in histone pre-mRNAs are discussed.

scholarly journals Reversion of protein aggregation mediated by Sso7d in cell extracts of Sulfolobus solfataricus

2004 ◽  
Vol 381 (1) ◽  
pp. 249-255 ◽  
Author(s):  
Annamaria GUAGLIARDI ◽  
Lucia MANCUSI ◽  
Mosè ROSSI
Keyword(s):  
Escherichia Coli ◽  
Protein Aggregation ◽  
Atp Hydrolysis ◽  
Sulfolobus Solfataricus ◽  
Protein Aggregates ◽  
Eukaryotic Cells ◽  
Cell Extracts ◽  
Thermophilic Archaeon

In eukaryotic cells and in Escherichia coli, reversion of protein aggregation is mediated by the network of chaperones belonging to Hsp70 and Hsp100 families [Weibezahn, Bukau and Mogk (2004) Microb. Cell Fact. 3, 1–12]. The thermophilic prokaryotes of the archaea domain lack homologues of these chaperone families, and the mechanisms they use to rescue aggregated proteins are unknown [Macario, Malz and Conway de Macario (2004) Front. Biosci. 9, 1318–1332]. In the present study, we show that stable protein aggregates can be detected in extracts of starved cells of the thermophilic archaeon Sulfolobus solfataricus, and that the protein Sso7d interacts with the aggregates and mediates the disassembly of the aggregates and the re-activation of insolubilized β-glycosidase in the presence of ATP hydrolysis. Furthermore, we report that heat-induced protein aggregates in extracts of exponential cells of S. solfataricus contain Sso7d that rescues insolubilized proteins in the presence of ATP hydrolysis. Results of these experiments performed in cell extracts are consistent with an in vivo role of Sso7d in reverting protein aggregation.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

scholarly journals The growth response of rice (Oryza sativa L. var. FARO 44) in vitro after inoculation with bacterial isolates from a typical ferruginous ultisol

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Musa Saheed Ibrahim ◽  
Beckley Ikhajiagbe
Keyword(s):  
16S Rrna ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Growth Response ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Rrna Gene Sequencing

Abstract Background Rice forms a significant portion of food consumed in most household worldwide. Rice production has been hampered by soil factors such as ferruginousity which has limited phosphorus availability; an important mineral component for the growth and yield of rice. The presence of phosphate-solubilizing bacteria (PSB) in soils has been reported to enhance phosphate availability. In view of this, the present study employed three bacteria species (BCAC2, EMBF2 and BCAF1) that were previously isolated and proved P solubilization capacities as inocula to investigate the growth response of rice germinants in an in vitro setup. The bacteria isolates were first identified using 16S rRNA gene sequencing and then applied as inoculum. The inolula were prepared in three concentrations (10, 7.5 and 5.0 ml) following McFarland standard. Viable rice (var. FARO 44) seeds were sown in petri dishes and then inoculated with the three inocula at the different concentrations. The setup was studied for 28 days. Results 16S rRNA gene sequencing identified the isolates as: isolate BCAC2= Bacillus cereus strain GGBSU-1, isolate BCAF1= Proteus mirabilis strain TL14-1 and isolate EMBF2= Klebsiella variicola strain AUH-KAM-9. Significant improvement in rice germination, morphology, physiology and biomass parameters in the bacteria-inoculated setups was observed compared to the control. Germination percentage after 4 days was 100 % in the inoculated rice germinants compared to 65% in the control (NiS). Similarly, inoculation with the test isolates enhanced water-use efficiency by over 40%. The rice seedlings inoculated with Bacillus cereus strain GGBSU-1 (BiS) showed no signs of chlorosis and necrosis throughout the study period as against those inoculated with Proteus mirabilis strain TL14-1 (PiS) and Klebsiella variicola strain AUH-KAM-9 (KiS). Significant increase in chlorophyll-a, chlorophyll-b and alpha amylase was observed in the rice seedlings inoculated with BiS as against the NiS. Conclusion Inoculating rice seeds with Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 in an in vitro media significantly improved growth parameters of the test plant. Bacillus cereus strain GGBSU-1 showed higher efficiency due to a more improved growth properties observed.

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