scholarly journals Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Virtu Solano-Collado ◽  
Sofía Ruiz-Cruz ◽  
Fabián Lorenzo-Díaz ◽  
Radoslaw Pluta ◽  
Manuel Espinosa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulation Of Gene Expression ◽  
E Coli ◽  
Core Enzyme ◽  
Protein Architecture ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

scholarly journals Regulation of Gene Expression of phiEco32-like Bacteriophage 7-11

2022 ◽  
Author(s):  
Daria Lavysh ◽  
Vladimir Mekler ◽  
Evgeny Klimuk ◽  
Konstantin Severinov
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Regulation Of Gene Expression ◽  
In Vitro Transcription ◽  
Single Element ◽  
Bacterial Rna ◽  
Sigma 70 ◽  
Rna Polymerase Holoenzyme ◽  
Rna Polymerase Promoter

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity  subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11  subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36  factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

scholarly journals Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1

2009 ◽  
Vol 191 (12) ◽  
pp. 3763-3771 ◽  
Author(s):  
Mohammed Dehbi ◽  
Gregory Moeck ◽  
Francis F. Arhin ◽  
Pascale Bauda ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Growth Inhibitory ◽  
Σ Factor ◽  
Transcription In Vitro

ABSTRACT The primary sigma factor of Staphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSA both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSA that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSA to DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

scholarly journals Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Russell K. Karls ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroidescytochrome c 2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterizedEscherichia coli heat shock (ς32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coliEς32 −35 consensus sequence (G34T) increasedcycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς38) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).

scholarly journals Crl Facilitates RNA Polymerase Holoenzyme Formation

2006 ◽  
Vol 188 (22) ◽  
pp. 7966-7970 ◽  
Author(s):  
Tamas Gaal ◽  
Mark J. Mandel ◽  
Thomas J. Silhavy ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Mechanism Of Action ◽  
Sigma Factor ◽  
Transcriptional Coactivator ◽  
Transcription System ◽  
The Core ◽  
Core Enzyme ◽  
Rna Polymerase Holoenzyme

ABSTRACT The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor σS. In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the EσS RNA polymerase holoenzyme but also by Eσ70 and Eσ32. Crl increased transcription dramatically but only when the σ concentration was low and when Crl was added to σ prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.

scholarly journals Mutagenesis of Region 4 of Sigma 28 from Chlamydia trachomatis Defines Determinants for Protein-Protein and Protein-DNA Interactions

2008 ◽  
Vol 191 (2) ◽  
pp. 651-660 ◽  
Author(s):  
Ziyu Hua ◽  
Xiancai Rao ◽  
Xiaogeng Feng ◽  
Xudong Luo ◽  
Yanmei Liang ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Morphological Differentiation ◽  
Amino Acid Residues ◽  
E Coli ◽  
Promoter Recognition ◽  
Protein Dna Interactions ◽  
Serovar Typhimurium ◽  
Insight Into

ABSTRACT Transcription factor σ28 in Chlamydia trachomatis (σ28 Ct) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in Salmonella enterica serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of σ28 Ct that were defective in σ28-specific transcription. Specially, the previously undefined but important interactions between σ28 Ct region 4 and the flap domain of the RNA polymerase β subunit (β-flap) or the −35 element of the chlamydial hctB promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of σ28 Ct contribute to an interaction with the β-flap when σ28 Ct associates with the core RNA polymerase. These residues function in contacts with the β-flap similarly to their counterpart residues in Escherichia coli σ70. Conversely, residue Q236 of σ28 Ct directly binds the chlamydial hctB −35 element. The conserved counterpart residue in E. coli σ70 has not been reported to interact with the −35 element of the σ70 promoter. Observed functional disparity between σ28 Ct and σ70 region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by σ28 Ct in C. trachomatis.

scholarly journals Transcription Activation In Vitro by the Bradyrhizobium japonicum Regulatory Protein FixK2

2005 ◽  
Vol 187 (10) ◽  
pp. 3329-3338 ◽  
Author(s):  
Socorro Mesa ◽  
Zöhre Ucurum ◽  
Hauke Hennecke ◽  
Hans-Martin Fischer
Keyword(s):  
Rna Polymerase ◽  
Bradyrhizobium Japonicum ◽  
Regulatory Protein ◽  
Transcription Activation ◽  
Receptor Protein ◽  
Nucleotide Position ◽  
Class Iii ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT In Bradyrhizobium japonicum, the N2-fixing root nodule endosymbiont of soybean, a group of genes required for microaerobic, anaerobic, or symbiotic growth is controlled by FixK2, a key regulator that is part of the FixLJ-FixK2 cascade. FixK2 belongs to the family of cyclic AMP receptor protein/fumarate and nitrate reductase (CRP/FNR) transcription factors that recognize a palindromic DNA motif (CRP/FNR box) associated with the regulated promoters. Here, we report on a biochemical analysis of FixK2 and its transcription activation activity in vitro. FixK2 was expressed in Escherichia coli and purified as a soluble N-terminally histidine-tagged protein. Gel filtration experiments revealed that increasing the protein concentration shifts the monomer-dimer equilibrium toward the dimer. Purified FixK2 productively interacted with the B. japonicum σ80-RNA polymerase holoenzyme, but not with E. coli σ70-RNA polymerase holoenzyme, to activate transcription from the B. japonicum fixNOQP, fixGHIS, and hemN 2 promoters in vitro. Furthermore, FixK2 activated transcription from the E. coli FF(−41.5) model promoter, again only in concert with B. japonicum RNA polymerase. All of these promoters are so-called class II CRP/FNR-type promoters. We showed by specific mutagenesis that the FixK2 box at nucleotide position −40.5 in the hemN 2 promoter, but not that at −78.5, is crucial for activation both in vivo and in vitro, which argues against recognition of a potential class III promoter. Given the lack of any evidence for the presence of a cofactor in purified FixK2, we surmise that FixK2 alone is sufficient to activate in vitro transcription to at least a basal level. This contrasts with all well-studied CRP/FNR-type proteins, which do require coregulators.

scholarly journals Trehalose Is Not Relevant for In Vivo Activity of ςS-Containing RNA Polymerase in Escherichia coli

1998 ◽  
Vol 180 (6) ◽  
pp. 1603-1606 ◽  
Author(s):  
Jens Germer ◽  
Andrea Muffler ◽  
Regine Hengge-Aronis
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Wild Type Strain ◽  
In Vitro Assays ◽  
Wild Type ◽  
Core Enzyme ◽  
Stress Response Genes ◽  
Promoter Recognition

ABSTRACT The ςS- and ς70-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on ςS. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of EςS and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649–3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various ςS-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of ςS-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.

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