scholarly journals 3′-immature tRNATrp is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase

1995 ◽  
Vol 310 (1) ◽  
pp. 249-253 ◽  
Author(s):  
M Brigotti ◽  
D Carnicelli ◽  
P Alvergna ◽  
A Pallanca ◽  
R Lorenzetti ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Liver Extract ◽  
Ribonuclease H ◽  
Ribosome Inactivating Protein ◽  
Mobility Shift ◽  
Glycosidase Activity ◽  
Ribosome Inactivation ◽  
Gel Mobility Shift ◽  
High Level ◽  
Good Acceptor

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3′-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.

SoxRS-mediated regulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1707-1716 ◽  
Author(s):  
Dagmar Rother ◽  
Grazyna Orawski ◽  
Frank Bardischewsky ◽  
Cornelius G. Friedrich
Keyword(s):  
Sequence Analysis ◽  
Sulfur Oxidation ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Cysteine Residue ◽  
Mobility Shift ◽  
C Terminus ◽  
Intergenic Regions ◽  
Paracoccus Pantotrophus ◽  
Gel Mobility Shift

Paracoccus pantotrophus GB17 requires thiosulfate for induction of the sulfur-oxidizing (Sox) enzyme system. The soxRS genes are divergently oriented to the soxVWXYZA–H genes. soxR predicts a transcriptional regulator of the ArsR family and soxS a periplasmic thioredoxin. The homogenote mutant GBΩS carrying a disruption of soxS by the Ω-kanamycin-resistance-encoding interposon expressed a low thiosulfate-oxidizing activity under heterotrophic and mixotrophic growth conditions. This activity was repressed by complementation with soxR, suggesting that SoxR acts as a repressor and SoxS is essential for full expression. Sequence analysis uncovered operator characteristics in the intergenic regions soxS–soxV and soxW–soxX. In each region a transcription start site was identified by primer extension analysis. Both regions were cloned into the vector pRI1 and transferred to P. pantotrophus. Strains harbouring pRI1 with soxS–soxV or soxW–soxX expressed the sox genes under heterotrophic conditions at a low rate, indicating repressor titration. Sequence analysis of SoxR suggested a helix–turn–helix (HTH) motif at position 87–108 and uncovered an invariant Cys-80 and a cysteine residue at the C-terminus. SoxR was overproduced in Escherichia coli with an N-terminal His6-tag and purified to near homogeneity. Electrophoretic gel mobility shift assays with SoxR retarded the soxS–soxV region as a single band while the soxW–soxX region revealed at least two protein–DNA complexes. These data demonstrated binding of SoxR to the relevant DNA. This is believed to be the first report of regulation of chemotrophic sulfur oxidation at the molecular level.

A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene

1990 ◽  
Vol 10 (4) ◽  
pp. 1470-1475
Author(s):  
A Yanagida ◽  
K Sogawa ◽  
K I Yasumoto ◽  
Y Fujii-Kuriyama
Keyword(s):  
Regulatory Element ◽  
Consensus Sequence ◽  
Inducible Expression ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Tata Sequence ◽  
Gel Mobility Shift ◽  
High Level

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

scholarly journals Regulation of Transcription of themph(A) Gene for Macrolide 2′-Phosphotransferase I inEscherichia coli: Characterization of the Regulatory Gene mphR(A)

2000 ◽  
Vol 182 (18) ◽  
pp. 5052-5058 ◽  
Author(s):  
Norihisa Noguchi ◽  
Katsutoshi Takada ◽  
Jin Katayama ◽  
Ayako Emura ◽  
Masanori Sasatsu
Keyword(s):  
Regulatory Gene ◽  
Regulation Of Gene Expression ◽  
Northern Blotting ◽  
Complementation Test ◽  
Regulation Of Transcription ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Α Helix ◽  
Gel Mobility Shift ◽  
High Level

ABSTRACT The synthesis of macrolide 2′-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires themph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied themphR(A) gene, which regulates the inducible expression ofmph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated thatmphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, andmphR(A) genes exist as a cluster that begins withmph(A) and that the deduced amino acid sequence of MphR(A) can adopt an α-helix–turn–α-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2.9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region ofmph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of themph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.

scholarly journals A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.

1990 ◽  
Vol 10 (4) ◽  
pp. 1470-1475 ◽  
Author(s):  
A Yanagida ◽  
K Sogawa ◽  
K I Yasumoto ◽  
Y Fujii-Kuriyama
Keyword(s):  
Regulatory Element ◽  
Consensus Sequence ◽  
Inducible Expression ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Tata Sequence ◽  
Gel Mobility Shift ◽  
High Level

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals FarRRegulates the farAB-Encoded Efflux Pump of Neisseriagonorrhoeae via an MtrR RegulatoryMechanism

2003 ◽  
Vol 185 (24) ◽  
pp. 7145-7152 ◽  
Author(s):  
E.-H. Lee ◽  
C. Rouquette-Loughlin ◽  
J. P. Folster ◽  
W. M. Shafer
Keyword(s):  
Regulatory Mechanism ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Transcriptional Repressors ◽  
Sequence Database ◽  
Mobility Shift ◽  
Pump System ◽  
Regulatory Cascade ◽  
Genome Sequence Database ◽  
Gel Mobility Shift

ABSTRACT The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

scholarly journals Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

2001 ◽  
Vol 45 (12) ◽  
pp. 3422-3426 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Tracy L. Twinem ◽  
Kelly M. Makin ◽  
Mark A. Nienaber ◽  
Chuiying Li ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Efflux Pump ◽  
Serial Passage ◽  
Efflux Pump Inhibitor ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
High Level ◽  
Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2861-2872 ◽  
Author(s):  
Marco Ventura ◽  
John G. Kenny ◽  
Ziding Zhang ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
3D Structure ◽  
Binding Mode ◽  
Dna Interaction ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Mobility Shift ◽  
Bifidobacterium Breve ◽  
Gel Mobility Shift ◽  
Slot Blot Analysis

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.

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