scholarly journals Autoregulator Protein PhaR for Biosynthesis of Polyhydroxybutyrate [P(3HB)] Possibly Has Two Separate Domains That Bind to the Target DNA and P(3HB): Functional Mapping of Amino Acid Residues Responsible for DNA Binding

2006 ◽  
Vol 189 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Miwa Yamada ◽  
Koichi Yamashita ◽  
Akiko Wakuda ◽  
Kazuyoshi Ichimura ◽  
Akira Maehara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Fluorescent Protein ◽  
Paracoccus Denitrificans ◽  
Regulatory Protein ◽  
Amino Acid Residues ◽  
Surface Plasmon Resonance Analysis ◽  
Target Dna ◽  
Green Fluorescent

ABSTRACT PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

scholarly journals Cd-Specific Mutants of Mercury-Sensing Regulatory Protein MerR, Generated by Directed Evolution

2011 ◽  
Vol 77 (17) ◽  
pp. 6215-6224 ◽  
Author(s):  
Kaisa M. Hakkila ◽  
Pia A. Nikander ◽  
Sini M. Junttila ◽  
Urpo J. Lamminmäki ◽  
Marko P. Virta
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Metal Binding ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Screening Strategy ◽  
Mercury Resistance ◽  
Content Type ◽  
Green Fluorescent ◽  
Mercury Sensing

ABSTRACTThe mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via themeroperator/promoter. AnEscherichia colicell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

scholarly journals Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

2006 ◽  
Vol 395 (3) ◽  
pp. 501-507 ◽  
Author(s):  
John Wilkinson ◽  
Xiumin Di ◽  
Kai Schönig ◽  
Joan L. Buss ◽  
Nancy D. Kock ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Protein Activity ◽  
Specific Expression ◽  
Cellular Iron ◽  
Tissue Iron ◽  
Green Fluorescent ◽  
Ferritin H

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTACMV (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTACMV transgenes, FerH expression was increased 6.0±1.1-fold (mean±S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2S-S2LAP (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2±4.8-fold (mean±S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3±0.9-fold (mean±S.D.), concurrent with a 4.5±2.1-fold (mean±S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.

scholarly journals Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2507
Author(s):  
Anja J. Engel ◽  
Laura-Marie Winterstein ◽  
Marina Kithil ◽  
Markus Langhans ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
K Channel ◽  
Physiological Changes ◽  
Channel Activity ◽  
Cryptochrome 2 ◽  
Green Fluorescent ◽  
Gfp Tag

The inner membranes of mitochondria contain several types of K+ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K+ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K+ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K+ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm2) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K+ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K+ conductance also results in a decrease in the Ca2+ concentration in the mitochondria but has no impact on apoptosis.

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

scholarly journals H-NS Silences gfp, the Green Fluorescent Protein Gene: gfpTCD Is a Genetically Remastered gfp Gene with Reduced Susceptibility to H-NS-Mediated Transcription Silencing and with Enhanced Translation

2010 ◽  
Vol 192 (18) ◽  
pp. 4790-4793 ◽  
Author(s):  
Colin P. Corcoran ◽  
Andrew D. S. Cameron ◽  
Charles J. Dorman
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Reporter Gene ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Bacterial Nucleoid ◽  
Green Fluorescent

ABSTRACT The bacterial nucleoid-associated protein H-NS, which preferentially targets and silences A+T-rich genes, binds the ubiquitous reporter gene gfp and dramatically reduces local transcription. We have redesigned gfp to reduce H-NS-mediated transcription silencing and simultaneously improve translation in vivo without altering the amino acid sequence of the GFP protein.

scholarly journals A Red Fluorescent Protein-Based Probe for Detection of Intracellular Reactive Sulfane Sulfur

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 985
Author(s):  
Zimai Li ◽  
Qingda Wang ◽  
Yongzhen Xia ◽  
Luying Xun ◽  
Huaiwei Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Cellular Component ◽  
Specific Probe ◽  
Amino Acid Residues ◽  
Red Fluorescent Protein ◽  
Sulfane Sulfur ◽  
Emission Ratio

Reactive sulfane sulfur, including persulfide and polysulfide, is a type of regular cellular component, playing an antioxidant role. Its function may be organelle-dependent; however, the shortage of probes for detecting organellar reactive sulfane sulfur has hindered further investigation. Herein, we reported a red fluorescent protein (mCherry)-based probe for specifically detecting intracellular reactive sulfane sulfur. By mutating two amino acid residues of mCherry (A150 and S151) to cysteine residues, we constructed a mCherry mutant, which reacted with reactive sulfane sulfur to form an intramolecular –Sn– bond (n ≥ 3). The bond largely decreased the intensity of 610 nm emission (excitation at 587 nm) and slightly increased the intensity of 466 nm emission (excitation at 406 nm). The 466/610 nm emission ratio was used to indicate the relative abundance of reactive sulfane sulfur. We then expressed this mutant in the cytoplasm and mitochondria of Saccharomyces cerevisiae. The 466/610 nm emission ratio revealed that mitochondria had a higher level of reactive sulfane sulfur than cytoplasm. Thus, the mCherry mutant can be used as a specific probe for detecting reactive sulfane sulfur in vivo.

scholarly journals Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

2004 ◽  
Vol 70 (6) ◽  
pp. 3263-3271 ◽  
Author(s):  
Anja Hoffmann ◽  
Tanja Schneider ◽  
Ulrike Pag ◽  
Hans-Georg Sahl
Keyword(s):  
Amino Acid ◽  
Staphylococcus Epidermidis ◽  
Fluorescent Protein ◽  
Terminal Segment ◽  
Terminal Part ◽  
C Terminus ◽  
Amino Acid Stretch ◽  
Green Fluorescent ◽  
Immunity Mechanism

ABSTRACT Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.

scholarly journals Μελέτη της σχέσης δομή - λειτουργία του κύριου μεταφορέα προλίνης (PrnB) στο μύκητα Aspergillus nidulans

2002 ◽  
Author(s):  
Στέφανος Ταβουλάρης
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Transmembrane Segments ◽  
Green Fluorescent

Το γονίδιο prnB του μύκητα Aspergillus nidulans κωδικοποιεί ένα ειδικό για την προλίνη μεταφορέα. Ο μεταφορέας αυτός είναι μια διαμεμβρανική πρωτεΐνη με κυτταροπλασματικό αμινοτελικό και καρβοξυτελικό άκρο, που αποτελείται από 12 πιθανά διαμεμβρανικά τμήματα α-έλικας (TMS: Transmembrane segments), τα οποία συνδέονται μεταξύ τους με μικρές υδρόφιλες θηλειές (L: loops). Η πρωτεΐνη PrnB ανήκει στην οικογένεια μεταφορέων APC (Amino Acid Polyamine Organocation) μέλη της οποίας εντοπίζονται σε προκαρυωτικούς και ευκαρυωτικούς οργανισμούς. Στην παρούσα διδακτορική διατριβή μελετήθηκαν οι σχέσεις δομής-λειτουργίας του μεταφορέα PrnB μέσω της γενετικής, μοριακής και βιοχημικής μελέτης στελεχών του Α. nidulans που εκφράζουν μεταλλαγμένες πρωτεΐνες PrnB. Οι εν λόγω μεταλλαγές είτε απομονώθηκαν γενετικά είτε κατασκευάσθηκαν με in vitro μεταλλαξιγένεση. Τα αποτελέσματα έδειξαν ότι οι παρερμηνεύσιμες μεταλλαγές που επηρεάζουν άμεσα ή έμμεσα τη λειτουργία της πρωτεΐνης PrnB εντοπίζονται στα L2-TMS3 (prnB6, prnB144), TMS6 (prnBKL, prnBKE, prnB119), L6-TMS7 (prnB81), L8-TMS9-L9 (prnB117, prnB206, prnB508) και L10-TMS11 (prnB411) τμήματά της. Από τις παραπάνω μεταλλαγές ιδιαίτερο ρόλο στη λειτουργία της πρωτεΐνης PrnB φαίνεται να διαδραματίζουν τα αμινοξέα Κ245 και F248 του TMS6. Η Κ245 είναι ένα από τα δύο θετικά φορτισμένα αμινοξέα που εντοπίζονται μέσα σε διαμεμβρανικά τμήματα της PrnB ενώ η F248 είναι αυστηρά συντηρημένη σε όλα τα μέλη της οικογένειας APC που είναι καταχωρημένα στην τράπεζα δεδομένων SWISS-PROT. Αλλαγή των αμινοξέων αυτών προκαλεί σημαντική μείωση της χημικής συγγένειας της PrnB για την προλίνη. Επιπλέον, σημαντικό ρόλο στη λειτουργία της PrnB φαίνεται να έχει η περιοχή L8-TMS9, η οποία αντιστοιχεί στη σημαντική για τη λειτουργία μεταφορέων αμινοξέων συναινετική αμφιπαθική περιοχή CAR (Consensus Amphipathic Region). Διπλασιασμός 2 και 3 αμινοξέων στην εν λόγω περιοχή προκαλεί πλήρη απώλεια λειτουργίας και σημαντική μεταβολή των τιμών Km και Vmax της PrnB, αντίστοιχα. Ενδιαφέρον αποτελεί το γεγονός ότι αντίστοιχα με τα παραπάνω τμήματα άλλων μελών της οικογένειας μεταφορέων APC έχει δειχθεί ότι είναι σημαντικά για τη λειτουργία των μεταφορέων αυτών. Εν κατακλείδι, τα παραπάνω αποτελέσματα υποδεικνύουν ότι οι περιοχές TMS6 και L8-TMS9 του μεταφορέα PrnB αποτελούν μέρος της οδού πρόσδεσης ή/και μεταφοράς της προλίνης διαμέσω της κυτταρικής μεμβράνης. Στα πλαίσια εμβάθυνσης των σχέσεων δομής - λειτουργίας του μεταφορέα PrnB καθώς και για τη μελέτη της in vivo τοπογένεσής του χρησιμοποιήθηκε η πρωτεΐνη GFP (Green Fluorescent Protein). Ένας αριθμός χιμαιρικών γονιδίων prnB - gfp κατασκευάσθηκαν, ενσωματώθηκαν στο γενετικό τόπο του γονιδίου prnB, εκφράστηκαν από το φυσιολογικό παρακινητή του, και τα αντίστοιχα χιμαιρικά μόρια PrnB - GFP εντοπίστηκαν in vivo. Τα χιμαιρικά αυτά μόρια φέρουν 2, 4 ή 8 συνδετικά αμινοξέα μεταξύ των πρωτεϊνών PrnB και GFP. Τα εν λόγω αμινοξέα δεν τροποποιούν τη δευτεροταγή δομή των χιμαιρικών μορίων, καθώς επιλέχθηκαν να μην είναι συμβατά με το σχηματισμό στοιχείων δευτεροταγούς δομής. Βρέθηκε ότι μόνο τα χιμαιρικά μόρια με 4 συνδετικά αμινοξέα είναι πλήρως λειτουργικά στους 25°C (φυσιολογική θερμοκρασία ανάπτυξης του μύκητα). Τα αποτελέσματα αυτά δείχνουν ότι ο αριθμός και το είδος των συνδετικών αμινοξέων μεταξύ των δύο μελών των χιμαιρικών μορίων είναι σημαντικός για τη "φυσιολογική" τοπογένεσή τους στην κυτταρική μεμβράνη. Επιπλέον, η χρήση της πρωτεΐνης GFP για τον in vivo εντοπισμό του διαμεμβρανικού μεταφορέα PrnB είναι δυνατή χωρίς να απαιτείται η υπερέκφραση του τελευταίου. Τέλος, η μέθοδος που αναπτύχθηκε για την κατασκευή χιμαιρικών PrnB-GFP μορίων δίνει τη δυνατότητα ενσωμάτωσης και έκφρασης οποιουδήποτε prnB- αλληλομόρφου. Αυτό έχει ως συνέπεια το διαχωρισμό των μεταλλαγών που επηρεάζουν άμεσα τη λειτουργία της πρωτεΐνης PrnB από αυτές που επηρεάζουν την τοπογένεσή της.

scholarly journals Amino Acid Residues in LuxR Critical for Its Mechanism of Transcriptional Activation during Quorum Sensing inVibrio fischeri

2001 ◽  
Vol 183 (1) ◽  
pp. 387-392 ◽  
Author(s):  
Amy E. Trott ◽  
Ann M. Stevens
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Quorum Sensing ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type

ABSTRACT PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR. This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the Vibrio fischeri lux operon during quorum sensing. The ability of the variant forms of LuxR to activate transcription of the lux operon was examined by using in vivo assays in recombinant Escherichia coli. Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR. Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type. An in vivo DNA binding-repression assay in recombinant E. coli was subsequently used to measure the ability of the variant forms of LuxR to bind to the lux box, the binding site of LuxR at thelux operon promoter. All eight LuxR variants found to affect cellular luciferase levels were unable to bind to thelux box. An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding. None of the alanine substitutions in LuxR affected activation of transcription of the lux operon without also affecting DNA binding. These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.

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