scholarly journals Identification of a Second Region of the Spo0A Response Regulator of Bacillus subtilis Required for Transcription Activation

2000 ◽  
Vol 182 (15) ◽  
pp. 4352-4355 ◽  
Author(s):  
Dean A. Rowe-Magnus ◽  
Martin J. Richer ◽  
George B. Spiegelman
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Response Regulator ◽  
Transcription Activation ◽  
Terminal Amino ◽  
Negative Phenotype

ABSTRACT Deletion of the 10 C-terminal amino acids of the Bacillus subtilis response regulator Spo0A or valine substitution at D258 and L260 resulted in a sporulation-negative phenotype and loss of in vivo activation of the spoIIG and spoIIA operon promoters. Repression of the abrB promoter was not affected by the mutations. In combination with the previously characterized mutation (A257V), the results identify amino acids at positions 257, 258, and 260 as being required for transcription activation by Spo0A.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

scholarly journals RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced in vivo by an anti-farnesyl cysteine antibody

2000 ◽  
Vol 97 (21) ◽  
pp. 11626-11631 ◽  
Author(s):  
R. Baron ◽  
E. Fourcade ◽  
I. Lajoie-Mazenc ◽  
C. Allal ◽  
B. Couderc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Terminal Amino ◽  
Carboxyl Terminal

scholarly journals Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo

1980 ◽  
Vol 188 (3) ◽  
pp. 705-713 ◽  
Author(s):  
G Livesey ◽  
P Lund
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Branched Chain Amino Acids ◽  
Rat Tissues ◽  
Arterial Blood ◽  
Branched Chain ◽  
Arteriovenous Difference

1. A procedure is described for the purification of leucine dehydrogenase (EC 1.4.1.9) from Bacillus subtilis. 2. The preparation is suitable for the quantitative assay of branched-chain amino acids and their 2-oxoacid analogues. 3. The content of total branched-chain 2-oxoacids in freeze-clamped liver, kidney, heart or mammary gland of fed rats is less than 5 nmol/g fresh wt. Higher amounts are present in skeletal muscle and arterial blood (25 +/- 4 nmol per g fresh wt., and 33 +/- 6 nmol per ml respectively; means +/- S.D. of 3 and 11 animals respectively). The values are not significantly affected by starvation for 24 h. 4. Arteriovenous difference measurements show that considerable amounts of branched-chain 2-oxoacids are released by skeletal muscle into the circulation and similar amounts are removed by the liver (about 1 mmol/24 h in a 400 g rat).

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

Des-serine-proline brain natriuretic peptide 3–32 in cardiorenal regulation

2007 ◽  
Vol 292 (2) ◽  
pp. R897-R901 ◽  
Author(s):  
Guido Boerrigter ◽  
Lisa C. Costello-Boerrigter ◽  
Gail J. Harty ◽  
Harald Lapp ◽  
John C. Burnett
Keyword(s):  
Amino Acids ◽  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Urinary Sodium Excretion ◽  
Urine Flow ◽  
Accelerated Degradation ◽  
Equimolar Dose ◽  
Terminal Amino ◽  
Physiologic Role

Brain natriuretic peptide (BNP 1–32) plays an important physiologic role in cardiorenal homeostasis. Recently, it has been reported that BNP 1–32 is rapidly cleaved by the ubiquitous enzyme dipeptidyl peptidase IV to BNP 3–32, which lacks the two NH2-terminal amino acids of BNP 1–32. The bioactivity of BNP 3–32 in cardiorenal regulation is unknown. We hypothesized that BNP 3–32 has reduced vasodilating and natriuretic bioactivity compared with BNP 1–32 in vivo. Synthetic human BNP 3–32 and BNP 1–32 were administered to eight anesthetized normal canines. After baseline measurements, BNP 1–32 at 30 ng·kg−1·min−1 was administered, followed by a washout, a postinfusion clearance, and a clearance with an equimolar dose of BNP 3–32. In four studies, the sequence of BNP 1–32 and BNP 3–32 infusion was reversed. Peptides were compared by analyzing the changes from the respective preinfusion clearance to the respective infusion clearance. * P < 0.05 between peptides. BNP 3–32, unlike BNP 1–32, did not decrease mean arterial pressure (0 ± 1 vs. −7 ± 2* mmHg, respectively) and did not increase renal blood flow (+12 ± 10 vs. +52 ± 10* ml/min). Effects on heart rate and cardiac output were similar. Urinary sodium excretion increased 128 ± 18 μeq/min with BNP 3–32 and 338 ± 40* μeq/min with BNP 1–32. Urine flow increased 1.1 ± 0.2 ml/min with BNP 3–32 and 2.8 ± 0.4* ml/min with BNP 1–32. Plasma BNP immunoreactivity was lower with BNP 3–32, suggesting accelerated degradation. In this study, BNP 3–32 showed reduced natriuresis and diuresis and a lack of vasodilating actions compared with BNP 1–32.

scholarly journals The Gal3p-Gal80p-Gal4p Transcription Switch of Yeast: Gal3p Destabilizes the Gal80p-Gal4p Complex in Response to Galactose and ATP

1999 ◽  
Vol 19 (11) ◽  
pp. 7828-7840 ◽  
Author(s):  
Alok Kumar Sil ◽  
Samina Alam ◽  
Ping Xin ◽  
Ly Ma ◽  
Melissa Morgan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Genetic Interaction ◽  
Transcription Activation ◽  
Full Length ◽  
Signal Transducer ◽  
Binding Peptide ◽  
Two Hybrid

ABSTRACT The Gal3, Gal80, and Gal4 proteins of Saccharomyces cerevisiae comprise a signal transducer that governs the galactose-inducible Gal4p-mediated transcription activation ofGAL regulon genes. In the absence of galactose, Gal80p binds to Gal4p and prohibits Gal4p from activating transcription, whereas in the presence of galactose, Gal3p binds to Gal80p and relieves its inhibition of Gal4p. We have found that immunoprecipitation of full-length Gal4p from yeast extracts coprecipitates less Gal80p in the presence than in the absence of Gal3p, galactose, and ATP. We have also found that retention of Gal80p by GSTG4AD (amino acids [aa] 768 to 881) is markedly reduced in the presence compared to the absence of Gal3p, galactose, and ATP. Consistent with these in vitro results, an in vivo two-hybrid genetic interaction between Gal80p and Gal4p (aa 768 to 881) was shown to be weaker in the presence than in the absence of Gal3p and galactose. These compiled results indicate that the binding of Gal3p to Gal80p results in destabilization of a Gal80p-Gal4p complex. The destabilization was markedly higher for complexes consisting of G4AD (aa 768 to 881) than for full-length Gal4p, suggesting that Gal80p relocated to a second site on full-length Gal4p. Congruent with the idea of a second site, we discovered a two-hybrid genetic interaction involving Gal80p and the region of Gal4p encompassing aa 225 to 797, a region of Gal4p linearly remote from the previously recognized Gal80p binding peptide within Gal4p aa 768 to 881.

The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras

1988 ◽  
Vol 8 (9) ◽  
pp. 3960-3963
Author(s):  
J E Buss ◽  
C J Der ◽  
P A Solski
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Myristic Acid ◽  
Amino Terminus ◽  
Directed Mutagenesis ◽  
Ras Protein ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Crucial Part

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.

scholarly journals Sporulation Phenotype of a Bacillus subtilis Mutant Expressing an Unprocessable but Active σE Transcription Factor

2004 ◽  
Vol 186 (7) ◽  
pp. 1999-2005 ◽  
Author(s):  
Shonna McBride ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Stage Iii ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Proprotein Processing ◽  
Residual Inhibition

ABSTRACT σE, a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-σE) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus. A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature σE but is active without processing. In the present work, we investigated the sporulation defect in sigE335-expressing B. subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of σE activity that is responsible. Fluorescence microscopy demonstrated that sigE335-expressing B. subtilis progresses further into sporulation (stage III) than do strains lacking σE activity (stage II). Consistent with its stage III phenotype, and a defect in σE activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE). The Spo− phenotype of sigE335 was found to be recessive to wild-type sigE. In vivo assays of σE activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on σE335, still impairs its activity to function as a transcription factor. The data suggest that the 11-amino-acid extension on σE335 allows it to bind RNA polymerase and direct the resulting holoenzyme to σE-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter.

scholarly journals Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

2009 ◽  
Vol 75 (19) ◽  
pp. 6306-6311 ◽  
Author(s):  
Shota Atsumi ◽  
Zhen Li ◽  
James C. Liao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Acetolactate Synthase ◽  
Side Chains ◽  
Decarboxylase Activity ◽  
Small Side

ABSTRACTA pathway toward isobutanol production previously constructed inEscherichia coliinvolves 2-ketoacid decarboxylase (Kdc) fromLactococcus lactisthat decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase fromBacillus subtilis(AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.

scholarly journals Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis

2004 ◽  
Vol 186 (4) ◽  
pp. 1120-1128 ◽  
Author(s):  
K. A. Susanna ◽  
A. F. van der Werff ◽  
C. D. den Hengst ◽  
B. Calles ◽  
M. Salas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Complex Signal ◽  
Type I ◽  
Binding Characteristics ◽  
Α Subunits

ABSTRACT The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

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