scholarly journals DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

2015 ◽  
Vol 112 (50) ◽  
pp. E6862-E6871 ◽  
Author(s):  
Andrey Parshin ◽  
Anthony L. Shiver ◽  
Jookyung Lee ◽  
Maria Ozerova ◽  
Dina Schneidman-Duhovny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Amino Acid Starvation ◽  
Cross Linking ◽  
Molecular Function ◽  
Secondary Channel ◽  
Global Response ◽  
Microbial Life ◽  
Active Site Region

Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo–cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.

scholarly journals The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

An energy charge sensor for balancing RNA polymerase recycling and hibernation

2020 ◽  
Author(s):  
Markus Wahl ◽  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo Chen ◽  
Yong-Heng Huang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Energy Levels ◽  
Energy Charge ◽  
Coiled Coil ◽  
Main Channel ◽  
Rna Polymerase I ◽  
Secondary Channel ◽  
Polymerase I

Abstract Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNA polymerase secondary channel, dismantling the active site and displacing RNA; a unique helical protrusion inserts into the main channel, prying β and β’ subunits apart and dislodging DNA, aided by the δ subunit. HelD release depends on ATP, and a dimeric structure resembling hibernating RNA polymerase I suggests that HelD can induce dormancy at low energy levels. Our results reveal an ingenious mechanism by which active RNA polymerase pools are adjusted in response to the nutritional state.

scholarly journals The Non-Coding B2 RNA Binds to the DNA Cleft and Active-Site Region of RNA Polymerase II

2013 ◽  
Vol 425 (19) ◽  
pp. 3625-3638 ◽  
Author(s):  
Steven L. Ponicsan ◽  
Stephane Houel ◽  
William M. Old ◽  
Natalie G. Ahn ◽  
James A. Goodrich ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
B2 Rna ◽  
Active Site Region

scholarly journals TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

2017 ◽  
Vol 114 (28) ◽  
pp. E5539-E5548 ◽  
Author(s):  
Saumya Gopalkrishnan ◽  
Wilma Ross ◽  
Albert Y. Chen ◽  
Richard L. Gourse
Keyword(s):  
Active Site ◽  
Transcription Initiation ◽  
Coiled Coil ◽  
Global Regulator ◽  
Secondary Channel ◽  
Global Regulators ◽  
Extrachromosomal Elements ◽  
Distant Homolog ◽  
Active Site Region

TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

scholarly journals Escherichia coli DksA Binds to Free RNA Polymerase with Higher Affinity than to RNA Polymerase in an Open Complex

2009 ◽  
Vol 191 (18) ◽  
pp. 5854-5858 ◽  
Author(s):  
Christopher W. Lennon ◽  
Tamas Gaal ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Binding Affinity ◽  
Quantitative Assay ◽  
Secondary Channel ◽  
Apparent Affinity ◽  
Transcriptional Output

ABSTRACT The transcription factor DksA binds in the secondary channel of RNA polymerase (RNAP) and alters transcriptional output without interacting with DNA. Here we present a quantitative assay for measuring DksA binding affinity and illustrate its utility by determining the relative affinities of DksA for three different forms of RNAP. Whereas the apparent affinities of DksA for RNAP core and holoenzyme are the same, the apparent affinity of DksA for RNAP decreases almost 10-fold in an open complex. These results suggest that the conformation of RNAP present in an open complex is not optimal for DksA binding and that DNA directly or indirectly alters the interface between the two proteins.

scholarly journals Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

2006 ◽  
Vol 282 (7) ◽  
pp. 4553-4560 ◽  
Author(s):  
Kayo Koide ◽  
Saki Maegawa ◽  
Koreaki Ito ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Lipid Phase ◽  
Intramembrane Proteolysis ◽  
Regulated Intramembrane Proteolysis ◽  
Eukaryotic Organisms ◽  
Alkylating Reagents ◽  
Active Site Region

Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown. Here we studied the environment surrounding the active site region of RseP, an Escherichia coli S2P ortholog involved in the σE pathway of extracytoplasmic stress responses. RseP has two presumed active site motifs, HEXXH and LDG, located in membrane-cytoplasm boundary regions. We examined the reactivity of cysteine residues introduced within or in the vicinity of these two active site motifs with membrane-impermeable thiol-alkylating reagents under various conditions. The active site positions were inaccessible to the reagents in the native state, but many of them became partially modifiable in the presence of a chaotrope, while requiring simultaneous addition of a chaotrope and a detergent for full modification. These results suggest that the active site of RseP is not totally embedded in the lipid phase but located within a proteinaceous structure that is partially exposed to the aqueous milieu.

scholarly journals Synthesis of ribonucleic acid in purine-deficient Escherichia coli and a comparison with the effects of amino acid starvation

1970 ◽  
Vol 120 (1) ◽  
pp. 125-132 ◽  
Author(s):  
N. F. Varney ◽  
Gillian A. Thomas ◽  
K. Burton
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Rna Synthesis ◽  
Adenine Nucleotides ◽  
Amino Acid Starvation ◽  
Guanine Nucleotides ◽  
Purine Nucleotides ◽  
Acid Control

1. Experiments with rifampicin and stringent strains of Escherichia coli (pro−purB−rel+) indicate that purine deficiency does not decrease and may considerably increase the potential for RNA synthesis by RNA polymerase molecules that are bound to DNA and have already commenced transcription. 2. DNA–RNA hybridization experiments indicate that purine starvation increases the distribution of bound RNA polymerase molecules between the cistrons for mRNA and those for stable RNA. 3. Synthesis of β-galactosidase mRNA is more dependent on the ability to synthesize guanine nucleotides than on the ability to synthesize adenine nucleotides. 4. Amino acid starvation tends to decrease the potential for RNA synthesis by RNA polymerase molecules bound to DNA. 5. Since this effect differs from that due to purine starvation, amino acid control of RNA synthesis does not appear to operate solely by causing a deficiency of purine nucleotides. 6. The results are discussed in terms of the ability to initiate RNA chains and to extend them under different circumstances.

Sign in / Sign up

Export Citation Format

Share Document