scholarly journals Translational Attenuation Mechanism of ErmB Induction by Erythromycin Is Dependent on Two Leader Peptides

2021 ◽  
Vol 12 ◽  
Author(s):  
Shasha Wang ◽  
Kai Jiang ◽  
Xinyue Du ◽  
Yanli Lu ◽  
Lijun Liao ◽  
...  
Keyword(s):  
Premature Termination ◽  
Regulatory Region ◽  
Terminal Region ◽  
Leader Peptide ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Major Mechanism ◽  
Attenuation Mechanism

Ribosome stalling on ermBL at the tenth codon (Asp) is believed to be a major mechanism of ermB induction by erythromycin (Ery). In this study, we demonstrated that the mechanism of ermB induction by Ery depends not only on ermBL expression but also on previously unreported ermBL2 expression. Introducing premature termination codons in ermBL, we proved that translation of the N-terminal region of ermBL is the key component for ermB induced by Ery, whereas translation of the C-terminal region of ermBL did not affect Ery-induced ermB. Mutation of the tenth codon (Asp10) of ermBL with other amino acids showed that the degree of induction in vivo was not completely consistent with the data from the in vitro toe printing assay. Alanine-scanning mutagenesis of ermBL demonstrated that both N-terminal residues (R7-K11) and the latter part of ermBL (K20-K27) are critical for Ery induction of ermB. The frameshifting reporter plasmid showed that a new leader peptide, ermBL2, exists in the ermB regulatory region. Further, introducing premature termination mutation and alanine-scanning mutagenesis of ermBL2 demonstrated that the N-terminus of ermBL2 is essential for induction by Ery. Therefore, the detailed function of ermBL2 requires further study.

scholarly journals Mutations in the C-Terminal Region of TraM Provide Evidence for In Vivo TraM-TraD Interactions during F-Plasmid Conjugation

2005 ◽  
Vol 187 (14) ◽  
pp. 4767-4773 ◽  
Author(s):  
Jun Lu ◽  
Laura S. Frost
Keyword(s):  
Dna Binding ◽  
Stop Codon ◽  
Terminal Region ◽  
In Vitro Assays ◽  
F Plasmid ◽  
Bacterial Populations ◽  
Major Mechanism ◽  
Mating Signal

ABSTRACT Conjugation is a major mechanism for disseminating genetic information in bacterial populations, but the signal that triggers it is poorly understood in gram-negative bacteria. F-plasmid-mediated conjugation requires TraM, a homotetramer, which binds cooperatively to three binding sites within the origin of transfer. Using in vitro assays, TraM has previously been shown to interact with the coupling protein TraD. Here we present evidence that F conjugation also requires TraM-TraD interactions in vivo. A three-plasmid system was used to select mutations in TraM that are defective for F conjugation but competent for tetramerization and cooperative DNA binding to the traM promoter region. One mutation, K99E, was particularly defective in conjugation and was further characterized by affinity chromatography and coimmunoprecipitation assays that suggested it was defective in interacting with TraD. A C-terminal deletion (S79*, where the asterisk represents a stop codon) and a missense mutation (F121S), which affects tetramerization, also reduced the affinity of TraM for TraD. We propose that the C-terminal region of TraM interacts with TraD, whereas its N-terminal domain is involved in DNA binding. This arrangement of functional domains could in part allow TraM to receive the mating signal generated by donor-recipient contact and transfer it to the relaxosome, thereby triggering DNA transfer.

scholarly journals Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3

2004 ◽  
Vol 186 (5) ◽  
pp. 1493-1502 ◽  
Author(s):  
Yinghua Chen ◽  
Wael R. Abdel-Fattah ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Phosphate Deficiency ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Α Helix

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoPH205A and PhoPV204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoPV202A and PhoPD203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoPV190A, PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoPW191A and PhoPR200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli.

scholarly journals Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.

1995 ◽  
Vol 15 (3) ◽  
pp. 1234-1243 ◽  
Author(s):  
J J Kang ◽  
D T Auble ◽  
J A Ranish ◽  
S Hahn
Keyword(s):  
Transcription Factor ◽  
Large Subunit ◽  
Small Subunit ◽  
Specific Factor ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Pol Ii ◽  
Conserved Regions

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

1993 ◽  
Vol 13 (9) ◽  
pp. 5710-5724
Author(s):  
E DesJardins ◽  
N Hay
Keyword(s):  
Zinc Finger ◽  
Tandem Repeats ◽  
Zinc Finger Protein ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Maximal Activity ◽  
Single Copy ◽  
Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

scholarly journals Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ivy Aneas ◽  
Donna C. Decker ◽  
Chanie L. Howard ◽  
Débora R. Sobreira ◽  
Noboru J. Sakabe ◽  
...  
Keyword(s):  
Association Studies ◽  
Regulatory Region ◽  
Airway Epithelial Cells ◽  
Genome Wide Association Studies ◽  
Airway Epithelial ◽  
Allele Specific ◽  
Enhancer Blocking ◽  
Underlying Mechanisms

AbstractGenome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

scholarly journals A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

2004 ◽  
Vol 24 (21) ◽  
pp. 9568-9579 ◽  
Author(s):  
Yanjiao Zhou ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Terminal Region ◽  
Replication Complex ◽  
Dna Polymerase Α ◽  
Chromatid Cohesion ◽  
Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region

1995 ◽  
Vol 108 (4) ◽  
pp. 1779-1789 ◽  
Author(s):  
K.C. Chang ◽  
K. Fernandes ◽  
M.J. Dauncey
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Chain Gene ◽  
Specific Expression ◽  
Slow Fibre ◽  
Skeletal Myosin

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation

1987 ◽  
Vol 7 (9) ◽  
pp. 3252-3259
Author(s):  
T Prezant ◽  
K Pfeifer ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Binding Studies ◽  
Multiple Factors ◽  
Vitro Binding ◽  
Genes Encoding ◽  
Nonfermentable Carbon Source

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

scholarly journals Transcriptional Regulation of ZNF638 in Thermogenic Cells by the cAMP Response Element Binding Protein in Male Mice

2019 ◽  
Vol 3 (12) ◽  
pp. 2326-2340 ◽  
Author(s):  
Luce Perie ◽  
Narendra Verma ◽  
Lingyan Xu ◽  
Xinran Ma ◽  
Elisabetta Mueller
Keyword(s):  
Zinc Finger ◽  
Binding Protein ◽  
Regulatory Region ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Response Element ◽  
Element Binding Protein ◽  
Thermogenic Adipocytes

Abstract Zinc finger factors are implicated in a variety of cellular processes, including adipose tissue differentiation and thermogenesis. We have previously demonstrated that zinc finger protein 638 (ZNF638) is a transcriptional coactivator acting as an early regulator of adipogenesis in vitro. In this study, we show, to our knowledge for the first time, that, in vivo, ZNF638 abounds selectively in mature brown and subcutaneous fat tissues and in fully differentiated thermogenic adipocytes. Furthermore, gene expression studies revealed that ZNF638 is upregulated by cAMP modulators in vitro and by cold exposure and by pharmacological stimulation of β-adrenergic signaling in vivo. In silico analysis of the upstream regulatory region of the ZNF638 gene identified two putative cAMP response elements within 500 bp of the ZNF638 transcription start site. Detailed molecular analysis involving EMSA and chromatin immunoprecipitation assays demonstrated that cAMP response element binding protein (CREB) binds to these cAMP response element regions of the ZNF638 promoter, and functional studies revealed that CREB is necessary and sufficient to regulate the levels of ZNF638 transcripts. Taken together, these results demonstrate that ZNF638 is selectively expressed in mature thermogenic adipocytes and tissues and that its induction in response to classic stimuli that promote heat generation is mediated via CREB signaling, pointing to a possible novel role of ZNF638 in brown and beige fat tissues.

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