Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro

Bożena Bruhn-Olszewska, Vadim Molodtsov, Michał Sobala, Maciej Dylewski, Katsuhiko Murakami, Michael Cashel, Katarzyna Potrykus

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5′triphosphate-3′diphosphate and guanosine 3′ 5′-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5′ and 3′ moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.

Original languageEnglish (US)
Pages (from-to)731-742
Number of pages12
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume1861
Issue number8
DOIs
StatePublished - Aug 1 2018

Fingerprint

DNA-Directed RNA Polymerases
Escherichia coli
Binding Sites
Transcription
Guanosine
Yin-Yang
Diphosphates
Guanine
Gene Expression Regulation
Second Messenger Systems
Adenine
adenosine 3'-diphosphate 5'-diphosphate
In Vitro Techniques
Gene expression
Plasticity
Catalytic Domain
Bacteria
Nucleotides
Crystal structure
Switches

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Bruhn-Olszewska, Bożena ; Molodtsov, Vadim ; Sobala, Michał ; Dylewski, Maciej ; Murakami, Katsuhiko ; Cashel, Michael ; Potrykus, Katarzyna. / Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro. In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms. 2018 ; Vol. 1861, No. 8. pp. 731-742.
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Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro. / Bruhn-Olszewska, Bożena; Molodtsov, Vadim; Sobala, Michał; Dylewski, Maciej; Murakami, Katsuhiko; Cashel, Michael; Potrykus, Katarzyna.

In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, Vol. 1861, No. 8, 01.08.2018, p. 731-742.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro

AU - Bruhn-Olszewska, Bożena

AU - Molodtsov, Vadim

AU - Sobala, Michał

AU - Dylewski, Maciej

AU - Murakami, Katsuhiko

AU - Cashel, Michael

AU - Potrykus, Katarzyna

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N2 - Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5′triphosphate-3′diphosphate and guanosine 3′ 5′-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5′ and 3′ moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.

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