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 S. Murakami , Michael Cashel , Katarzyna Potrykus


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.
Author Bożena Bruhn-Olszewska KGMB
Bożena Bruhn-Olszewska,,
- Department of Molecular Genetics of Bacteria
, Vadim Molodtsov
Vadim Molodtsov,,
, Michał Sobala KGMB
Michał Sobala ,,
- Department of Molecular Genetics of Bacteria
, Maciej Dylewski KGMB
Maciej Dylewski ,,
- Department of Molecular Genetics of Bacteria
, Katsuhiko S. Murakami
Katsuhiko S. Murakami,,
, Michael Cashel
Michael Cashel,,
, Katarzyna Potrykus KGMB
Katarzyna Potrykus,,
- Department of Molecular Genetics of Bacteria
Journal seriesBiochimica et Biophysica Acta-Gene Regulatory Mechanisms, ISSN 1874-9399
Issue year2018
Publication size in sheets0.55
Keywords in EnglishRNAP, transcription, rrnB P1, ppGpp, (p)ppGpp, (p)ppApp
Languageen angielski
Score (nominal)40
ScoreMinisterial score = 40.0, 23-07-2018, ArticleFromJournal
Ministerial score (2013-2016) = 40.0, 23-07-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 5.018 (2) - 2016=5.373 (5)
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.