Identification of Rhodococcus erythropolis Promoters Controlled by Alternative Sigma Factors Using In Vivo and In Vitro Systems and Heterologous RNA Polymerase

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

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ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

Journal of Bacteriology ◽

10.1128/jb.182.16.4606-4616.2000 ◽

2000 ◽

Vol 182 (16) ◽

pp. 4606-4616 ◽

Cited By ~ 96

Author(s):

Maureen J. Bibb ◽

Virginie Molle ◽

Mark J. Buttner

Keyword(s):

Rna Polymerase ◽

Sigma Factor ◽

Streptomyces Coelicolor ◽

Aerial Mycelium ◽

Sigma Factors ◽

Colony Development ◽

Null Mutant ◽

Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

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Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Journal of Bacteriology ◽

10.1128/jb.01792-07 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3434-3443 ◽

Cited By ~ 24

Author(s):

Umender K. Sharma ◽

Dipankar Chatterji

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Rna Polymerase ◽

Bacteriophage T4 ◽

Sigma Factors ◽

E Coli ◽

Yeast Two Hybrid System ◽

Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

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A Complex Network of Sigma Factors and sRNA StsR Regulates Stress Responses in R. sphaeroides

International Journal of Molecular Sciences ◽

10.3390/ijms22147557 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7557

Author(s):

Katrin M. H. Eisenhardt ◽

Bernhardt Remes ◽

Julian Grützner ◽

Daniel-Timon Spanka ◽

Andreas Jäger ◽

...

Keyword(s):

Stress Responses ◽

Sigma Factors ◽

Photooxidative Stress ◽

Alternative Sigma Factors ◽

Special Challenge ◽

Photosynthesis Genes ◽

In Vitro Interaction ◽

Complex Regulatory Network

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.

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Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

Biochemistry and Cell Biology ◽

10.1139/o05-061 ◽

2005 ◽

Vol 83 (4) ◽

pp. 497-504 ◽

Cited By ~ 2

Author(s):

Benoit Coulombe ◽

Marie-France Langelier

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Catalytic Mechanism ◽

Mutational Analysis ◽

Structural Elements ◽

Catalytic Mechanisms ◽

Rnap Ii ◽

Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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Human Oligodendrocytes and Myelin In Vitro to Evaluate Developmental Neurotoxicity

International Journal of Molecular Sciences ◽

10.3390/ijms22157929 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7929

Author(s):

Megan Chesnut ◽

Thomas Hartung ◽

Helena Hogberg ◽

David Pamies

Keyword(s):

Large Scale ◽

In Vitro Models ◽

Environmental Chemicals ◽

Developmental Neurotoxicity ◽

In Vivo Studies ◽

Regulatory Guidelines ◽

In Vitro Systems ◽

Human Oligodendrocytes

Neurodevelopment is uniquely sensitive to toxic insults and there are concerns that environmental chemicals are contributing to widespread subclinical developmental neurotoxicity (DNT). Increased DNT evaluation is needed due to the lack of such information for most chemicals in common use, but in vivo studies recommended in regulatory guidelines are not practical for the large-scale screening of potential DNT chemicals. It is widely acknowledged that developmental neurotoxicity is a consequence of disruptions to basic processes in neurodevelopment and that testing strategies using human cell-based in vitro systems that mimic these processes could aid in prioritizing chemicals with DNT potential. Myelination is a fundamental process in neurodevelopment that should be included in a DNT testing strategy, but there are very few in vitro models of myelination. Thus, there is a need to establish an in vitro myelination assay for DNT. Here, we summarize the routes of myelin toxicity and the known models to study this particular endpoint.

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Protein-templated gold nanoclusters as specific bio-imaging probes for the detection of Hg(ii) ions in in vivo and in vitro systems: discriminating between MDA-MB-231 and MCF10A cells

The Analyst ◽

10.1039/d0an02108c ◽

2021 ◽

Author(s):

Subhajit Chakraborty ◽

Atanu Nandy ◽

Subhadip Ghosh ◽

Nirmal Kumar Das ◽

Sameena Parveen ◽

...

Keyword(s):

Breast Cancer ◽

Human Serum Albumin ◽

Gold Nanoclusters ◽

Selective Detection ◽

Imaging Probes ◽

Bio Imaging ◽

In Vitro Systems ◽

Mcf10a Cells

Sub-nanomolar selective detection of Hg(ii) ions by protein (Human Serum Albumin, HSA) templated gold nanoclusters (AuNCs), both in in vitro as well as in vivo environments and specific endocytose behaviour towards breast cancer (BC) cell lines.

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Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

Journal of Biological Chemistry ◽

10.1074/jbc.m009634200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12266-12273 ◽

Cited By ~ 31

Author(s):

Wenxiang Wei ◽

Dorjbal Dorjsuren ◽

Yong Lin ◽

Weiping Qin ◽

Takahiro Nomura ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Middle Part ◽

Wild Type ◽

Binding Region ◽

Pol Ii ◽

Transcription Factor Iif ◽

B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603271113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E2899-E2905 ◽

Cited By ~ 23

Author(s):

Irina O. Vvedenskaya ◽

Hanif Vahedian-Movahed ◽

Yuanchao Zhang ◽

Deanne M. Taylor ◽

Richard H. Ebright ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Transcription Initiation ◽

Specific Protein ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433-5441.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

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