Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription

ABSTRACT Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rho-dependent terminator. A comparison of terminator read-through in a wild-type Escherichia coli strain and that in a nusB::IS10 mutant strain determined the requirement for NusB. In the absence of NusB, antiterminator-dependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusG-depleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.

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Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00159-18 ◽

2018 ◽

Vol 200 (12) ◽

Author(s):

Chunyou Mao ◽

Yan Zhu ◽

Pei Lu ◽

Lipeng Feng ◽

Shiyun Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Essential Role ◽

Β Subunit ◽

Content Type ◽

E Coli ◽

Core Enzyme ◽

Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

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Subunit assembly in vivo of Escherichia coli RNA polymerase: role of the amino‐terminal assembly domain of alpha subunit

Genes to Cells ◽

10.1046/j.1365-2443.1996.d01-258.x ◽

1996 ◽

Vol 1 (6) ◽

pp. 517-528 ◽

Cited By ~ 14

Author(s):

Makoto Kimura ◽

Akira Ishihama

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Alpha Subunit ◽

Subunit Assembly ◽

Amino Terminal

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The Escherichia coli Regulator of Sigma 70 Protein, Rsd, Can Up-Regulate Some Stress-Dependent Promoters by Sequestering Sigma 70

Journal of Bacteriology ◽

10.1128/jb.00019-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3489-3495 ◽

Cited By ~ 30

Author(s):

Jennie E. Mitchell ◽

Taku Oshima ◽

Sarah E. Piper ◽

Christine L. Webster ◽

Lars F. Westblade ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Hybrid System ◽

Transcriptome Analysis ◽

Biological Role ◽

Stress Dependent ◽

Sigma 70 ◽

Two Hybrid ◽

Stimulation Of

ABSTRACT The Escherichia coli Rsd protein forms complexes with the RNA polymerase σ70 factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative σ38 factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with σ70. Our experiments support a model in which the role of Rsd is primarily to sequester σ70, thereby increasing the levels of RNA polymerase containing the alternative σ38 factor.

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UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker

Nucleic Acids Research ◽

10.1093/nar/29.20.4166 ◽

2001 ◽

Vol 29 (20) ◽

pp. 4166-4178 ◽

Cited By ~ 29

Author(s):

W. Meng

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Alpha Subunit

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The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts

mBio ◽

10.1128/mbio.00184-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Deani L. Cooper ◽

Taku Harada ◽

Samia Tamazi ◽

Alexander E. Ferrazzoli ◽

Susan T. Lovett

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Rna Polymerase ◽

Transcription Elongation ◽

Clamp Loader ◽

Content Type ◽

Growth Defects ◽

Transcriptional Termination ◽

Transcription Complexes

ABSTRACT In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β), and RpoC (β′) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity. IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC’s absence.

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