Chemical nuclease activity of 5-phenyl-1,10-phenanthroline-copper ion detects intermediates in transcription initiation by E. Coli RNA polymerase

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

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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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Use of Fast DNA Footprinting and Real-Time Fluorescence Spectroscopy to Characterize Novel, Biologically Revlevant Transcription Initiation Intermediates for E. Coli RNA Polymerase

Biophysical Journal ◽

10.1016/j.bpj.2011.11.1581 ◽

2012 ◽

Vol 102 (3) ◽

pp. 286a

Author(s):

Sara Heitkamp ◽

Emily Lingeman ◽

Mike Capp ◽

Ruth Saecker ◽

M. Thomas Record

Keyword(s):

Fluorescence Spectroscopy ◽

Rna Polymerase ◽

Real Time ◽

Transcription Initiation ◽

E Coli ◽

Dna Footprinting

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E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

10.1101/766725 ◽

2019 ◽

Author(s):

James Chen ◽

Saumya Gopalkrishnan ◽

Courtney Chiu ◽

Albert Y. Chen ◽

Elizabeth A. Campbell ◽

...

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Transcription Initiation ◽

Structural Changes ◽

E Coli ◽

Bacterial Proteins ◽

Cryo Electron Microscopy ◽

Promoter Complex ◽

Promoter Dna ◽

Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

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Abortive and Productive Transcription Initiation by E. coli RNA Polymerase

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1372 ◽

2018 ◽

Vol 114 (3) ◽

pp. 246a-247a

Author(s):

Kate Henderson ◽

Cristen M. Molzahn ◽

Lindsey C. Felth ◽

Claire Evensen ◽

Sarah Dyke ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

E Coli

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Decision letter: E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

10.7554/elife.49375.sa1 ◽

2019 ◽

Author(s):

Dong Wang

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

E Coli

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

Journal of Bacteriology ◽

10.1128/jb.180.1.1-9.1998 ◽

1998 ◽

Vol 180 (1) ◽

pp. 1-9 ◽

Cited By ~ 8

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).

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