scholarly journals Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

2015 ◽  
Vol 197 (7) ◽  
pp. 1197-1207 ◽  
Author(s):  
Guangze Jin ◽  
Martin S. Pavelka ◽  
J. Scott Butler
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Function Analysis ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Binding Domain ◽  
Type Ii ◽  
Toxin Binding ◽  
Dna Binding Specificity ◽  
Amino Acid Side Chains

ABSTRACTBacterial toxin-antitoxin systems play a critical role in the regulation of gene expression, leading to developmental changes, reversible dormancy, and cell death. Type II toxin-antitoxin pairs, composed of protein toxins and antitoxins, exist in nearly all bacteria and are classified into six groups on the basis of the structure of the toxins. The VapBC group comprises the most common type II system and, like other toxin-antitoxin systems, functions to elicit dormancy by inhibiting protein synthesis. Activation of toxin function requires protease degradation of the VapB antitoxin, which frees the VapC toxin from the VapBC complex, allowing it to hydrolyze the RNAs required for translation. Generally, type II antitoxins bind with high specificity to their cognate toxins via a toxin-binding domain and endow the complex with DNA-binding specificity via a DNA-binding domain. Despite the ubiquity of VapBC systems and their critical role in the regulation of gene expression, few functional studies have addressed the details of VapB-VapC interactions. Here we report on the results of experiments designed to identify molecular determinants of the specificity of theMycobacterium tuberculosisVapB4 antitoxin for its cognate VapC4 toxin. The results identify the minimal domain of VapB4 required for this interaction as well as the amino acid side chains required for binding to VapC4. These findings have important implications for the evolution of VapBC toxin-antitoxin systems and their potential as targets of small-molecule protein-protein interaction inhibitors.IMPORTANCEVapBC toxin-antitoxin pairs are the most widespread type II toxin-antitoxin systems in bacteria, where they are thought to play key roles in stress-induced dormancy and the formation of persisters. The VapB antitoxins are critical to these processes because they inhibit the activity of the toxins and provide the DNA-binding specificity that controls the synthesis of both proteins. Despite the importance of VapB antitoxins and the existence of several VapBC crystal structures, little is known about their functional featuresin vivo. Here we report the findings of the first comprehensive structure-function analysis of a VapB toxin. The results identify the minimal toxin-binding domain, its modular antitoxin function, and the specific amino acid side chains required for its activity.

The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity

1991 ◽  
Vol 11 (8) ◽  
pp. 3925-3930
Author(s):  
C L Dent ◽  
K A Lillycrop ◽  
J K Estridge ◽  
N S Thomas ◽  
D S Latchman
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
B Cell ◽  
Functional Activity ◽  
Critical Role ◽  
Neuronal Cell ◽  
Binding Specificity ◽  
Cell Protein ◽  
Mobility Shift ◽  
Dna Binding Specificity

B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.

scholarly journals The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity.

1991 ◽  
Vol 11 (8) ◽  
pp. 3925-3930 ◽  
Author(s):  
C L Dent ◽  
K A Lillycrop ◽  
J K Estridge ◽  
N S Thomas ◽  
D S Latchman
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
B Cell ◽  
Functional Activity ◽  
Critical Role ◽  
Neuronal Cell ◽  
Binding Specificity ◽  
Cell Protein ◽  
Mobility Shift ◽  
Dna Binding Specificity

B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Regulation of type II adenylyl cyclase mRNA in rabbit skeletal muscle by chronic motor nerve pacing

1996 ◽  
Vol 271 (2) ◽  
pp. E253-E260 ◽  
Author(s):  
C. E. Torgan ◽  
W. E. Kraus
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Regulation Of Gene Expression ◽  
Rabbit Skeletal Muscle ◽  
Type Ii ◽  
Wide Range ◽  
Electrical Pacing ◽  
Fast Twitch

Skeletal muscle exhibits a wide range in functional phenotype in response to changes in physiological demands. We have observed that, in response to changes in work patterns, alterations in gene expression of some proteins coincide with changes in adenylyl cyclase (AC) activity [Kraus, W.E., J.P. Longabaugh, and S. B. Liggett. Am. J. Physiol 263 (Endocrinol. Metab. 26): E266-E230, 1992]. We now examine AC isoform transcript prevalence in various rabbit skeletal muscles and in response to changing work demands. Using reverse transcriptase-polymerase chain reaction, we detected type II AC isoform transcripts in rabbit skeletal muscle. Ribonuclease protection analyses revealed that expression of the type II isoform significantly correlated with the percentage of fast-twitch type IIb/IId fibers (r2 = 0.765, P < 0.01). When a fast-twitch muscle was converted to a slow-twitch muscle via chronic electrical pacing, expression of type II AC mRNA significantly decreased. This response occurred 3 days after the onset of stimulation (78% decrease) and was still present after 21 days of stimulation (76% decrease). As type II AC is relatively insensitive to calcium regulation while sensitive to protein kinase C (PKC) signaling, these data provide further impetus for investigations of protein kinase A and PKC cross-talk signaling mechanisms in the regulation of gene expression.

Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain

Cell ◽  
1988 ◽  
Vol 52 (5) ◽  
pp. 685-695 ◽  
Author(s):  
Stuart Adler ◽  
Marian L. Waterman ◽  
Xi He ◽  
Michael G. Rosenfeld
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Steroid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Prolactin Gene

scholarly journals Adaptive evolution of transcription factor binding affinities

2017 ◽  
Author(s):  
Jungeui Hong ◽  
Nathan Brandt ◽  
Ally Yang ◽  
Tim Hughes ◽  
David Gresham
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Adaptive Evolution ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Missense Mutations ◽  
Binding Affinities ◽  
Synonymous Mutations

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

scholarly journals AguR Is Required for Induction of the Streptococcus mutans Agmatine Deiminase System by Low pH and Agmatine

2009 ◽  
Vol 75 (9) ◽  
pp. 2629-2637 ◽  
Author(s):  
Yaling Liu ◽  
Lin Zeng ◽  
Robert A. Burne
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Streptococcus Mutans ◽  
Low Ph ◽  
Binding Domain ◽  
Negative Regulator ◽  
Dna Binding Domain ◽  
Mutant Proteins ◽  
Acidic Conditions ◽  
Agmatine Deiminase

ABSTRACT Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of Streptococcus mutans. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated aguR genes that were utilized to complement an aguR deletion mutant of S. mutans. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the agu operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.

scholarly journals ADAR1 Interacts with NF90 through Double-Stranded RNA and Regulates NF90-Mediated Gene Expression Independently of RNA Editing

2005 ◽  
Vol 25 (16) ◽  
pp. 6956-6963 ◽  
Author(s):  
Yongzhan Nie ◽  
Li Ding ◽  
Peter N. Kao ◽  
Robert Braun ◽  
Jing-Hua Yang
Keyword(s):  
Gene Expression ◽  
Rna Editing ◽  
Nuclear Export ◽  
Fetal Liver ◽  
Regulation Of Gene Expression ◽  
Nuclear Export Signal ◽  
Binding Domain ◽  
Double Stranded Rna ◽  
Recognition Element ◽  
Dsrna Binding

ABSTRACT The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 was recently demonstrated to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. Here we report a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins.

Chromatin-Modifying Enzymes in T Cell Development

2020 ◽  
Vol 38 (1) ◽  
pp. 397-419
Author(s):  
Michael J. Shapiro ◽  
Virginia Smith Shapiro
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Biological Effects ◽  
Critical Role ◽  
T Cell Development ◽  
Regulation Of Gene Expression ◽  
Chromatin Accessibility ◽  
Cell Development ◽  
Specific Gene ◽  
Dynamic Regulation

T cell development involves stepwise progression through defined stages that give rise to multiple T cell subtypes, and this is accompanied by the establishment of stage-specific gene expression. Changes in chromatin accessibility and chromatin modifications accompany changes in gene expression during T cell development. Chromatin-modifying enzymes that add or reverse covalent modifications to DNA and histones have a critical role in the dynamic regulation of gene expression throughout T cell development. As each chromatin-modifying enzyme has multiple family members that are typically all coexpressed during T cell development, their function is sometimes revealed only when two related enzymes are concurrently deleted. This work has also revealed that the biological effects of these enzymes often involve regulation of a limited set of targets. The growing diversity in the types and sites of modification, as well as the potential for a single enzyme to catalyze multiple modifications, is also highlighted.

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