Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from <i>Bacillus subtilis</i>

Papoušková, Veronika; Kadeřávek, Pavel; Otrusinová, Olga; Rabatinová, Alžběta; Šanderová, Hana; Nováček, Jiří; Krásný, Libor; Sklenář, Vladimı́r; Žı́dek, Lukáš

doi:10.1002/cbic.201300226

Cited by 19 publications

(20 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It consists of an amino-terminal globular domain and a nucleic acid-mimicking highly acidic unstructured C-terminal half. 47,48 The δ protein forms a complex with the RNAP core enzyme in a 1:1 stoichiometry. 42,47,49 A truncated form of δ lacking the C-terminal half is sufficient for binding to RNAP.…”

Section: Resultsmentioning

confidence: 99%

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

Jong¹,

Koning²,

Roseboom³

et al. 2017

J. Proteome Res.

View full text Add to dashboard Cite

Identification of dynamic protein–protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.

show abstract

Section: Resultsmentioning

confidence: 99%

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

Jong¹,

Koning²,

Roseboom³

et al. 2017

J. Proteome Res.

View full text Add to dashboard Cite

show abstract

“…When one compares RpoE protein sequences between various Gram-positive bacteria, there does appear to be a large variation in similarity other than in regions of the collected N termini. The N-terminal regions are characterized by the presence of several ␣-helices and ␤-sheets that were previously identified in the B. subtilis protein (28,31) and are predicted to be present in the S. aureus counterpart (www.predictprotein.org). Such conservation is perhaps to be expected, since the N terminus is thought to be the component that mediates interaction with core RNA polymerase (9), which is itself a highly conserved protein complex.…”

Section: Discussionmentioning

confidence: 99%

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

et al. 2014

View full text Add to dashboard Cite

In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the ␦ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the ␤ and/or ␤= subunits. To identify the impact of the ␦ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the ␦ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. Bacterial gene transcription is a complex, multifactorial process that involves several key enzymes and regulatory elements. It is driven by the activity of DNA-dependent RNA polymerase (RNAP) and its associated proteins, which form a multisubunit enzyme consisting of one ␤ subunit, one ␤= subunit, two identical ␣ subunits, and one subunit (reviewed in reference 1). Together these form the RNAP apoenzyme, which is able to perform RNA elongation and termination; however, initiation requires the involvement of a factor. Typically, most bacterial species harbor several different factors, a primary one ( A or 70 ) that mediates housekeeping gene transcription and a variety of alternative sigma factors, which aid in the response to unfavorable environmental conditions and stress.In certain Gram-positive species and particularly the Firmicutes (see Fig. S1 in the supplemental material), an additional RNAP subunit is present, termed the ␦ factor, or RpoE (2). In Bacillus subtilis, the 173-amino-acid delta subunit has been shown to reduce nonspecific binding of RNAP to DNA and lead to an elevated preference for DNA regions that include promoter sequences (3, 4). In early experiments, it was shown that RpoE has a role specifically confined to promoter selection and influences the ability of RNAP to form open promoter complexes (5, 6). Fulllength RpoE has also been described as displacing nucleic acids from RNAP-DNA or RNAP-RNA complexes in vitro, which may explain the decreased affinity of RNAP-RpoE for nonpromoter regions. Thi...

show abstract

“…Taking into account that their interaction counterparts in salt bridges are positively charged amino acids with long flexible side chains (either Lys or Arg), such subtle variations are unlikely to affect the 1.1 function(s). The highest sequence conservation is around the HI-HII loop region (Leu 19 , Gly 23 , Lys 24 , Gly 27 , Thr 30 , and Tyr 31 ).…”

Section: Sequence Comparisonsmentioning

confidence: 99%

Solution structure of domain 1.1 of the σA factor from Bacillus subtilis is preformed for binding to the RNA polymerase core

Zachrdla

Padrta

Rabatinová

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from in complex with RNAP and from solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium We found that σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to σ1.1. Moreover, modeling studies suggested that σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas σ1.1 must be rearranged to fit therein. Thus, the mesophilic species and share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in , bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.

show abstract

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

Cited by 19 publications

References 33 publications

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

The δ Subunit of RNA Polymerase Guides Promoter Selectivity and Virulence in Staphylococcus aureus

Solution structure of domain 1.1 of the σA factor from Bacillus subtilis is preformed for binding to the RNA polymerase core

Contact Info

Product

Resources

About