scholarly journals Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Cierra A. Birch ◽  
Madison J. Davis ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Stress Induction ◽  
Content Type ◽  
Terminal Domain ◽  
Codon Substitution

ABSTRACT Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP α subunit (αCTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in αCTD severely impaired Spx-activated transcription. Attempts to substitute alanine for αCTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where “Y263C” indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound αCTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli αD259, which has been implicated in αCTD-σ70 interaction (σ70 R603, corresponding to R362 of B. subtilis σA). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while αCTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-αCTD complex. IMPORTANCE Though extensively studied in Escherichia coli, the role of αCTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis. Here, we conduct phenotypic analyses of putatively lethal αCTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-αCTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several αCTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

scholarly journals RNA polymerases in strict endosymbiont bacteria with extreme genome reduction show distinct erosions that might result in limited and differential promoter recognition

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0239350
Author(s):  
Cynthia Paola Rangel-Chávez ◽  
Edgardo Galán-Vásquez ◽  
Azucena Pescador-Tapia ◽  
Luis Delaye ◽  
Agustino Martínez-Antonio
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerases ◽  
Genome Reduction ◽  
Promoter Element ◽  
Promoter Elements ◽  
Α Subunit ◽  
Amino Terminal ◽  
Terminal Domain

Strict endosymbiont bacteria present high degree genome reduction, retain smaller proteins, and in some instances, lack complete functional domains compared to free-living counterparts. Until now, the mechanisms underlying these genetic reductions are not well understood. In this study, the conservation of RNA polymerases, the essential machinery for gene expression, is analyzed in endosymbiont bacteria with extreme genome reductions. We analyzed the RNA polymerase subunits to identify and define domains, subdomains, and specific amino acids involved in precise biological functions known in Escherichia coli. We also perform phylogenetic analysis and three-dimensional models over four lineages of endosymbiotic proteobacteria with the smallest genomes known to date: Candidatus Hodgkinia cicadicola, Candidatus Tremblaya phenacola, Candidatus Tremblaya Princeps, Candidatus Nasuia deltocephalinicola, and Candidatus Carsonella ruddii. We found that some Hodgkinia strains do not encode for the RNA polymerase α subunit. The rest encode genes for α, β, β’, and σ subunits to form the RNA polymerase. However, 16% shorter, on average, respect their orthologous in E. coli. In the α subunit, the amino-terminal domain is the most conserved. Regarding the β and β’ subunits, both the catalytic core and the assembly domains are the most conserved. However, they showed compensatory amino acid substitutions to adapt to changes in the σ subunit. Precisely, the most erosive diversity occurs within the σ subunit. We identified broad amino acid substitution even in those recognizing and binding to the -10-box promoter element. In an overall conceptual image, the RNA polymerase from Candidatus Nasuia conserved the highest similarity with Escherichia coli RNA polymerase and their σ70. It might be recognizing the two main promoter elements (-10 and -35) and the two promoter accessory elements (-10 extended and UP-element). In Candidatus Carsonella, the RNA polymerase could recognize all the promoter elements except the -10-box extended. In Candidatus Tremblaya and Hodgkinia, due to the α carboxyl-terminal domain absence, they might not recognize the UP-promoter element. We also identified the lack of the β flap-tip helix domain in most Hodgkinia’s that suggests the inability to bind the -35-box promoter element.

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

scholarly journals Crystal structure of the in vivo-assembled Bacillus subtilis Spx/RNA polymerase α subunit C-terminal domain complex

2009 ◽  
Vol 168 (2) ◽  
pp. 352-356 ◽  
Author(s):  
Valerie Lamour ◽  
Lars F. Westblade ◽  
Elizabeth A. Campbell ◽  
Seth A. Darst
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Α Subunit ◽  
Subunit C ◽  
Terminal Domain

scholarly journals The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

2016 ◽  
Vol 198 (23) ◽  
pp. 3186-3199 ◽  
Author(s):  
Amit Pathania ◽  
Arvind Kumar Gupta ◽  
Swati Dubey ◽  
Balasubramanian Gopal ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cytoplasmic Membrane ◽  
Basic Amino Acid ◽  
Intramolecular Interactions ◽  
Membrane Topology ◽  
Content Type ◽  
E Coli ◽  
Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

scholarly journals Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

2005 ◽  
Vol 187 (5) ◽  
pp. 1724-1731 ◽  
Author(s):  
K. Derek Weber ◽  
Owen D. Vincent ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Class Ii ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Side Chains ◽  
Alanine Scanning Mutagenesis ◽  
Α Subunit

ABSTRACT The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(−61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the α subunit (αCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that αCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of αCTD required for FNR activation is more extensive than previously observed.

scholarly journals Activation by NarL at the Escherichia coli ogt promoter

2020 ◽  
Vol 477 (15) ◽  
pp. 2807-2820
Author(s):  
Patcharawarin Ruanto ◽  
David L. Chismon ◽  
Joanne Hothersall ◽  
Rita E. Godfrey ◽  
David J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Response Regulator ◽  
Direct Interaction ◽  
Α Subunit ◽  
E Coli ◽  
Terminal Domain ◽  
Dna Alkyltransferase ◽  
Promoter Dna ◽  
Transcript Initiation

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

scholarly journals The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 5110-5117 ◽  
Author(s):  
Carla Y. Bonilla ◽  
Alan D. Grossman
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Dna Binding ◽  
Replication Initiation ◽  
High Rate ◽  
Nucleotide Exchange ◽  
Replicative Helicase ◽  
Content Type ◽  
Aaa Atpase ◽  
Replication Initiator

ABSTRACTDnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites.Bacillus subtilisDnaD is needed during replication initiation for assembly of the replicative helicase atoriCand during replication restart at stalled replication forks. DnaD associates with DnaA atoriCand at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulateB. subtilisDnaA also affect DNA binding, whereas much of the regulation ofEscherichia coliDnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange forB. subtilisDnaA was high and not affected by DnaD. The rapid exchange is similar to that ofStaphylococcus aureusDnaA and in contrast to the low exchange rate ofEscherichia coliDnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.

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