REGULATION OF THE put OPERON IN SALMONELLA TYPHIMURIUM: CHARACTERIZATION OF PROMOTER AND OPERATOR MUTATIONS

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 687-703
Author(s):  
Donald R Hahn ◽  
Stanley R Maloy
Keyword(s):  
Regulatory Region ◽  
Lacz Expression ◽  
Proline Utilization ◽  
Single Operator ◽  
Regulatory Mutations ◽  
Operon Regulation ◽  
In Trans ◽  
Operon Expression ◽  
Operator Site

ABSTRACT The two genes required for proline utilization by S. typhimurium form a divergent operon. Expression of the put operon is induced by proline and subject to catabolite repression. Genetic evidence suggests that putA protein autogenously represses transcription of the putA and putP genes. In order to establish the molecular mechanism of put operon regulation we isolated regulatory mutations in the put control region. These mutants were selected using two phenotypes: (1) the ability to degrade a toxic proline analogue, dehydroproline, due to overexpression of putA enzyme activity, or (2) overexpression of lacZ from put::Mud operon fusions. The effect of each mutation on transcription in both directions was determined by measuring lacZ expression from putA and putP operon fusions. These regulatory mutations were cis-dominant when the putA protein was provided in trans, and they map in a region between the two genes. The phenotypes of the mutants suggest that (1) the put regulatory region has a single operator site where the putA protein binds to repress transcription in both directions, and (2) the putA and putP promoters overlap.

scholarly journals Identification and Characterization of a DeoR-Specific Operator Sequence Essential for Induction ofdra-nupC-pdp Operon Expression in Bacillus subtilis

1999 ◽  
Vol 181 (6) ◽  
pp. 1719-1727 ◽  
Author(s):  
Xianmin Zeng ◽  
Hans H. Saxild
Keyword(s):  
Bacillus Subtilis ◽  
Control Region ◽  
Regulatory Region ◽  
Direct Repeat ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Operon Expression ◽  
Specific Operator

ABSTRACT The deoR gene located just upstream thedra-nupC-pdp operon of Bacillus subtilisencodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptionallacZ fusions. It was shown that all of thecis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position −60 to position −43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3′ half of the palindrome and was located between the −10 and −35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

scholarly journals CHARACTERIZATION OF THE OPERATOR SITES OF THE exu REGULON IN ESCHERICHIA COLI K-12 BY OPERATOR-CONSTITUTIVE MUTATIONS AND REPRESSOR TITRATION

Genetics ◽  
1983 ◽  
Vol 105 (4) ◽  
pp. 829-842
Author(s):  
Mireille Mata-Gilsinger ◽  
Paul Ritzenthaler ◽  
Carlos Blanco
Keyword(s):  
Escherichia Coli ◽  
Recombination Event ◽  
Regulatory Gene ◽  
Negative Control ◽  
The Other ◽  
Multiple Copies ◽  
Operon Expression ◽  
K 12 ◽  
Operator Site

ABSTRACT In Escherichia coli, the exu regulon of the hexuronate system involves the three exuT, uxaCA and uxaB operons and is under the negative control of the exuR regulatory gene product. The technique developed by Casadaban, Chou and Cohen was employed to construct two plasmids containing operon fusions in which the lactose genes were fused to the uxaCA and exuT operons. These fusions were transferred into the chromosome by a reciprocal recombination event, and the resulting strains were used for isolation of mutants defective in repression. Two types of operator-constitutive mutants were obtained: one specific for the uxaCA operon expression and the other affecting the exuT gene expression. This genetic evidence confirms that these two operons which are divergently transcribed each possess their own operator site.—The derepressed expression of the two exuT-lac and uxaCA-lac operons and the uxaB gene was also examined upon introduction of plasmids bearing various operators of the exu regulon. The results of testing exuR repressor titration by multiple copies of the exu operators allowed us to show a gradation in the affinity degrees for the three exu operators: uxaBo has the strongest affinity for the exuR repressor and uxaCo the weakest, although that of exuTo seems to be just slightly greater. This gradation may play a role in the control of the exu regulon expression.

scholarly journals A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

2021 ◽  
Vol 22 (14) ◽  
pp. 7390
Author(s):  
Nicole Wesch ◽  
Frank Löhr ◽  
Natalia Rogova ◽  
Volker Dötsch ◽  
Vladimir V. Rogov
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Effective Interaction ◽  
Regulatory Region ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Cellular Processes ◽  
Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

scholarly journals A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

1988 ◽  
Vol 8 (11) ◽  
pp. 4634-4641 ◽  
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Gene Fusion ◽  
Regulatory Gene ◽  
Regulatory Region ◽  
Tata Box ◽  
Homologous Gene ◽  
Lacz Gene ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Necessary And Sufficient ◽  
Utilization Pathway

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

scholarly journals Single-operator cholangioscopy for diagnosis of cholangioadenoma (bile duct adenoma) and its potential impact on surgical management

2018 ◽  
Vol 06 (11) ◽  
pp. E1312-E1316 ◽  
Author(s):  
John Eccles ◽  
Aducio Thiesen ◽  
Gurpal Sandha
Keyword(s):  
Bile Duct ◽  
Surgical Management ◽  
Liver Enzymes ◽  
Clinical Indication ◽  
Targeted Biopsy ◽  
Patient Demographics ◽  
Single Operator ◽  
Potential Impact ◽  
Per Oral

Abstract Background and study aims Cholangioadenoma is not recognized commonly and is often only diagnosed on surgical specimens. Direct per oral single-operator cholangioscopy (SOC) allows characterization of common bile duct (CBD) lesions through direct visualization and directed forceps biopsies with potential for impacting surgical management decisions. This is a retrospective review of all SOC cases diagnosed with cholangioadenoma. Patient demographics and outcomes were recorded. Three patients (all male), average age 68 years (range 62 – 76 years), were identified to have a cholangioadenoma. The clinical indication for SOC was deranged liver enzymes with a dilated CBD and a CBD abnormality identified on biliary imaging. The site of cholangioadenoma was proximal, mid and distal CBD, respectively. All patients had a successful SOC with targeted biopsy-proven diagnosis. One patient had a synchronous cholangiocarcinoma and underwent palliative stenting whereas the other two patients underwent appropriate curative resection based on cholangioadenoma location. We conclude that SOC is safe and effective for diagnosis of cholangioadenoma and has potential impact on decisions for surgical management.

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