scholarly journals Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

2000 ◽  
Vol 182 (19) ◽  
pp. 5539-5550 ◽  
Author(s):  
Paula S. Fritsch ◽  
Mark L. Urbanowski ◽  
George V. Stauffer
Keyword(s):  
Rna Polymerase ◽  
Random Mutagenesis ◽  
Complex Surface ◽  
Α Subunit ◽  
C Terminus ◽  
Distinct Cluster ◽  
In Vitro Reconstitution ◽  
The Difference ◽  
Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-subunit of RNA polymerase

2021 ◽  
Author(s):  
Helen Camakaris ◽  
Ji Yang ◽  
Tadashi Fujii ◽  
James Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Protein ◽  
Aromatic Amino Acids ◽  
Regulation Of Transcription ◽  
Plant Interactions ◽  
Α Subunit ◽  
Partial Suppression ◽  
K 12

A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of E. coli K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act - mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. TyrR Mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77 and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act - tyrR mutants were isolated in an rpoA + strain, adding positions R77, D97, K101, D118, R119, R121 and E141 to known residues, S95 and D103, and defining the ‘activation patch’ on the NTD of TyrR. These results support a model for activation of TyrR-regulated genes where the ‘activation patch’ on the TyrR NTD interacts with the ‘TyrR-specific patch’ on the αCTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed, and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants, as Activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene TyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence, and important plant interactions.

scholarly journals Subforms and In Vitro Reconstitution of the NAD-Reducing Hydrogenase of Alcaligenes eutrophus

1998 ◽  
Vol 180 (5) ◽  
pp. 1023-1029 ◽  
Author(s):  
Christian Massanz ◽  
Silke Schmidt ◽  
Bärbel Friedrich
Keyword(s):  
Alcaligenes Eutrophus ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Monomeric Form ◽  
Structural Genes ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Reducing Activity

The cytoplasmic, NAD-reducing hydrogenase (SH) of Alcaligenes eutrophus H16 is a heterotetrameric enzyme which contains several cofactors and undergoes a complex maturation during biogenesis. HoxH is the Ni-carrying subunit, and together with HoxY it forms the hydrogenase dimer. HoxF and HoxU represent the flavin-containing diaphorase moiety, which is closely related to NADH:ubiquinone oxidoreductase and mediates NADH oxidation. A variety of mutations were introduced into the four SH structural genes to obtain mutant enzymes composed of monomeric and dimeric forms. A deletion removing most ofhoxF, hoxU, and hoxY led to the expression of a HoxH monomer derivative which was proteolytically processed at the C terminus like the wild-type polypeptide. While the hydrogenase dimer, produced by a strain deleted of hoxF andhoxU, displayed H2-dependent dye-reducing activity, the monomeric form did not mediate the activation of H2, although nickel was incorporated into HoxH. Deletion ofhoxH and hoxY led to the production of HoxFU dimers which displayed NADH:oxidoreductase activity. Mixing the hydrogenase and the diaphorase moieties in vitro reconstituted the structure and catalytic function of the SH holoenzyme.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

scholarly journals Structural analysis of fungal pathogenicity-related casein kinase α subunit, Cka1, in the human fungal pathogen Cryptococcus neoformans

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Belinda X. Ong ◽  
Youngki Yoo ◽  
Myeong Gil Han ◽  
Jun Bae Park ◽  
Myung Kyung Choi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Kinase Assay ◽  
Α Subunit ◽  
Dynamic Architecture ◽  
Human Fungal Pathogen ◽  
Life Threatening ◽  
The Difference ◽  
Anti Fungal

Abstract CK2α is a constitutively active and highly conserved serine/threonine protein kinase that is involved in the regulation of key cellular metabolic pathways and associated with a variety of tumours and cancers. The most well-known CK2α inhibitor is the human clinical trial candidate CX-4945, which has recently shown to exhibit not only anti-cancer, but also anti-fungal properties. This prompted us to work on the CK2α orthologue, Cka1, from the pathogenic fungus Cryptococcus neoformans, which causes life-threatening systemic cryptococcosis and meningoencephalitis mainly in immunocompromised individuals. At present, treatment of cryptococcosis remains a challenge due to limited anti-cryptococcal therapeutic strategies. Hence, expanding therapeutic options for the treatment of the disease is highly clinically relevant. Herein, we report the structures of Cka1-AMPPNP-Mg2+ (2.40 Å) and Cka1-CX-4945 (2.09 Å). Structural comparisons of Cka1-AMPPNP-Mg2+ with other orthologues revealed the dynamic architecture of the N-lobe across species. This may explain for the difference in binding affinities and deviations in protein-inhibitor interactions between Cka1-CX-4945 and human CK2α-CX-4945. Supporting it, in vitro kinase assay demonstrated that CX-4945 inhibited human CK2α much more efficiently than Cka1. Our results provide structural insights into the design of more selective inhibitors against Cka1.

Expression of Distinct α Subunits of GABAA Receptor Regulates Inhibitory Synaptic Strength

2004 ◽  
Vol 92 (3) ◽  
pp. 1718-1727 ◽  
Author(s):  
Pavel I. Ortinski ◽  
Congyi Lu ◽  
Kentaroh Takagaki ◽  
Zhanyan Fu ◽  
Stefano Vicini
Keyword(s):  
Primary Cultures ◽  
Molecular Assembly ◽  
Inhibitory Synapses ◽  
Critical Determinant ◽  
Pharmacological Agents ◽  
Α Subunit ◽  
Subunit Expression ◽  
Α Subunits ◽  
In Cells

Distinct α subunit subtypes in the molecular assembly of GABAA receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and α1 subunit −/− mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of α2 and α3 subunits, the increase of α1 and α6 subunits expression at synapses, and changes in the action of selective α subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from −/− mice together with a decrease in the expression of α3 subunit. In contrast, the expression of α2 subunit at inhibitory synapses in −/− cells was significantly higher than in +/+ cells at DIV11-12. α5 subunit was not detected, and increased sensitivity to a selective α4/α6 subunit agonist suggests increased expression of extrasynaptic receptors in −/− mice. β2/β3 subunit expression and loreclezole sensitivity increased with development in +/+ but not in −/− cells, supporting the preferential association of the α1 with the β2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the +/+ and −/− groups until DIV11-12. Our results uncover a pattern of sequential expression of α subunits underlying the changes in functional efficacy of GABAergic networks with development.

scholarly journals Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814 ◽  
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4

2019 ◽  
Vol 21 (1) ◽  
pp. 135 ◽  
Author(s):  
Sergey A. Proshkin ◽  
Elena K. Shematorova ◽  
George V. Shpakovski
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Fetal Brain ◽  
Gene Promoters ◽  
Α Subunit ◽  
Promoter Recognition ◽  
Rnap Ii ◽  
Human Specific

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals A stromal region of cytochromeb6fsubunit IV is involved in the activation of the Stt7 kinase inChlamydomonas

2017 ◽  
Vol 114 (45) ◽  
pp. 12063-12068 ◽  
Author(s):  
Louis Dumas ◽  
Francesca Zito ◽  
Stéphanie Blangy ◽  
Pascaline Auroy ◽  
Xenie Johnson ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Direct Interaction ◽  
Chloroplast Transformation ◽  
Kinase Domain ◽  
State Transitions ◽  
Light Harvesting Complexes ◽  
Gene Coding ◽  
In Vitro Reconstitution

The cytochrome (cyt)b6fcomplex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cytb6factivates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplastpetDgene, coding for subunit IV of the cytb6fcomplex, and complementation of a ΔpetDhost strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cytb6fsubunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cytb6fand recombinant Stt7 kinase domain show that cytb6fenhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cytb6fcomplex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

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