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

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.

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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

Journal of Bacteriology ◽

10.1128/jb.00124-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 2

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.

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

Biochemical Journal ◽

10.1042/bj3370415 ◽

1999 ◽

Vol 337 (3) ◽

pp. 415-423 ◽

Cited By ~ 8

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.

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

Journal of Bacteriology ◽

10.1128/jb.187.5.1724-1731.2005 ◽

2005 ◽

Vol 187 (5) ◽

pp. 1724-1731 ◽

Cited By ~ 14

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.

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Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter.

Journal of Bacteriology ◽

10.1128/jb.179.19.6187-6191.1997 ◽

1997 ◽

Vol 179 (19) ◽

pp. 6187-6191 ◽

Cited By ~ 13

Author(s):

J Yang ◽

K Murakami ◽

H Camakaris ◽

N Fujita ◽

A Ishihama ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Rna Polymerase ◽

Alpha Subunit ◽

Amino Acid Residues ◽

Subunit C ◽

Terminal Domain ◽

Activation Of Transcription

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Activation by NarL at the Escherichia coli ogt promoter

Biochemical Journal ◽

10.1042/bcj20200408 ◽

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.

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Spacing requirements for interactions between the C-terminal domain of the α subunit of Escherichia coli RNA polymerase and the cAMP receptor protein

Biochemical Journal ◽

10.1042/bj3300413 ◽

1998 ◽

Vol 330 (1) ◽

pp. 413-420 ◽

Cited By ~ 8

Author(s):

S. Georgina LLOYD ◽

J. W. Stephen BUSBY ◽

J. Nigel SAVERY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Binding Site ◽

Transcription Initiation ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Α Subunit ◽

Synthetic Promoters ◽

Terminal Domain ◽

Camp Receptor

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase α subunit (αCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with αCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified α subunits was studied. The footprints show that αCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent αCTD from being contacted by CRP. Models to account for this are discussed.

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Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

Journal of Bacteriology ◽

10.1128/jb.184.8.2273-2280.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2273-2280 ◽

Cited By ~ 50

Author(s):

Nigel J. Savery ◽

Georgina S. Lloyd ◽

Stephen J. W. Busby ◽

Mark S. Thomas ◽

Richard H. Ebright ◽

...

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Rna Polymerase ◽

Class Ii ◽

Class I ◽

Receptor Protein ◽

Α Subunit ◽

Cyclic Amp Receptor Protein ◽

Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

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RNA polymerases in strict endosymbiont bacteria with extreme genome reduction show distinct erosions that could result in limited and differential promoters’ recognition

10.1101/2020.09.07.285775 ◽

2020 ◽

Author(s):

Cynthia Paola Rangel-Chávez ◽

Edgardo Galán-Vásquez ◽

Azucena Pescador-Tapia ◽

Luis Delaye ◽

Agustino Martínez-Antonio

Keyword(s):

Rna Polymerase ◽

Positive Selection ◽

Three Dimensional ◽

Random Mutagenesis ◽

Rna Polymerases ◽

Genome Reduction ◽

Α Subunit ◽

Vital Functions ◽

Dimensional Models ◽

Three Dimensional Models

AbstractStrict endosymbiont bacteria with high degree of genome reduction retain smaller proteins and, in certain cases, lack complete functional domains compared to their free-living counterparts. Until now, the mechanisms underlying these genetic reductions are not well understood. However, it is thought that, in order to compensate for gene reduction, somehow hosts take over those vital functions that endosymbionts cannot perform. In the present study, the conservation of RNA polymerases, the essential machinery for gene expression, is analysed in bacteria with extreme genome reductions. For this purpose, comparative genomics, phylogenetic analysis and three-dimensional models of RNA polymerase subunits were done over four lineages of endosymbiotic proteobacteria with the smallest genomes known to date. Amino acids under positive selection in the α subunit and loss of motifs in other subunits of RNA polymerase were observed. According to three-dimensional models, sites under positive selection might compensate the loss of motifs in α subunit. In addition, variations in the σ subunit were identified, some of them already studied in E. coli as a result of random mutagenesis. Amino acid changes in RNA polymerase suggest a possible modification in the binding specificity of the canonical −10 box (TATAAT) in some of these organisms. Furthermore, the β-flap helix domain is absent in some Hodgkinia strains, as observed in RNA pol II of Archaea, thus lacking the capacity to bind to the −35 box. Here, we propose several RNA polymerases models for endosymbiont bacteria with extremely reduced genomes. Evidence suggests that RNA polymerases of each endosymbiont bacteria follow a unique evolutionary path, without necessarily following the same path as a lineage, this is probably influenced by the intimate interactions sustained with other endosymbionts and its hosts.

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