The RNA polymerase gene specificity factor sigma 54 is required for homogeneous non-planktonic growth of uropathogenic Escherichia coli

The canonical function of a bacterial sigma factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the sigma 54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the sigma 54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. The genes that rely upon sigma 54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by sigma 54, is not yet exhaustive. By comparing the non-planktonic growth properties of prototypical enteropathogenic, uropathogenic and non-pathogenic Escherichia coli strains devoid of sigma 54, we uncovered sigma 54 as a determinant of homogenous non-planktonic growth specifically in the uropathogenic strain. Notably, bacteria devoid of individual activator ATPases of the sigma 54-RNAP do not phenocopy the sigma 54 mutant strain. It seems that sigma 54's role as a determinant of homogenous non-planktonic growth represents a putative non-canonical function of sigma 54 in regulating genetic information flow.

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A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36816-3 ◽

1994 ◽

Vol 269 (18) ◽

pp. 13179-13184

Author(s):

S.E. Aiyar ◽

J.D. Helmann ◽

P.L. deHaseth

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Double Stranded Dna

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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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Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3623 ◽

2003 ◽

Vol 50 (4) ◽

pp. 909-920 ◽

Cited By ~ 4

Author(s):

Iwona K Kolasa ◽

Tomasz Łoziński ◽

Kazimierz L Wierzchowski

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Specific Interaction ◽

Structural Data ◽

Structural Morphology ◽

Promoter Dna ◽

Sigma Subunit ◽

Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

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Structural basis of RNA polymerase III transcription repression by Maf1

10.1101/859132 ◽

2019 ◽

Author(s):

Matthias K. Vorländer ◽

Florence Baudin ◽

Robyn D. Moir ◽

René Wetzel ◽

Wim J. H. Hagen ◽

...

Keyword(s):

Rna Polymerase ◽

Binding Site ◽

Transcription Initiation ◽

Rna Polymerase Iii ◽

Structural Basis ◽

Metabolic Efficiency ◽

Transcription Repression ◽

Polymerase Iii ◽

Wide Range ◽

Pol Iii

ABSTRACTMaf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

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Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10975-10985.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10975-10985 ◽

Cited By ~ 45

Author(s):

Mohamed A. Ghazy ◽

Seth A. Brodie ◽

Michelle L. Ammerman ◽

Lynn M. Ziegler ◽

Alfred S. Ponticelli

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Primer Extension ◽

Site Directed Mutagenesis ◽

Protein Encoding ◽

Transcription Factor Iif ◽

Growth Properties ◽

Encoding Genes

ABSTRACT Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

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Affinity Isolation and I-DIRT Mass Spectrometric Analysis of the Escherichia coli O157:H7 Sakai RNA Polymerase Complex

Journal of Bacteriology ◽

10.1128/jb.01599-07 ◽

2007 ◽

Vol 190 (4) ◽

pp. 1284-1289 ◽

Cited By ~ 14

Author(s):

David J. Lee ◽

Stephen J. W. Busby ◽

Lars F. Westblade ◽

Brian T. Chait

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Laboratory Strain ◽

Effector Proteins ◽

Spectrometric Analysis ◽

Isolation Technique ◽

E Coli ◽

Affinity Isolation ◽

K 12

ABSTRACT Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the Escherichia coli K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic Escherichia coli strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although E. coli O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the “core” E. coli genome.

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Translational Regulation of the Escherichia coli Stress Factor RpoS: a Role for SsrA and Lon

Journal of Bacteriology ◽

10.1128/jb.01838-06 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4872-4879 ◽

Cited By ~ 27

Author(s):

Caroline Ranquet ◽

Susan Gottesman

Keyword(s):

Escherichia Coli ◽

Low Temperature ◽

Rna Polymerase ◽

Transcription Initiation ◽

Translational Regulation ◽

Regulatory Rna ◽

Gene Encoding ◽

Ribosome Stalling ◽

High Level ◽

Sigma Subunit

ABSTRACT Escherichia coli cell viability during starvation is strongly dependent on the expression of the rpoS gene, encoding the RpoS sigma subunit of RNA polymerase. RpoS abundance has been reported to be regulated at many levels, including transcription initiation, translation, and protein stability. The regulatory RNA SsrA (or tmRNA) has both tRNA and mRNA activities, relieving ribosome stalling and cotranslationally tagging proteins. We report here that SsrA is needed for the correct high-level translation of RpoS. The ATP-dependent protease Lon was also found to negatively affect RpoS translation, but only at low temperature. We suggest that SsrA may indirectly improve RpoS translation by limiting ribosome stalling and depletion of some component of the translation machinery.

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A Novel Initiation Pathway in Escherichia Coli Transcription

10.1101/042432 ◽

2016 ◽

Cited By ~ 2

Author(s):

Eitan Lerner ◽

SangYoon Chung ◽

Benjamin L. Allen ◽

Shuang Wang ◽

Jookyung J. Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Rate Limiting Step ◽

Limiting Step ◽

Magnetic Tweezer ◽

Delayed Initiation ◽

Rate Limiting ◽

Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

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