Crl activates transcription by stabilizing the active conformation of the master stress factor σS

SUMMARYσS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses and antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an E. coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNAP holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with the domain 2 of σS (σS2), sharing no interaction with promoter DNA. Subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) results indicate that Crl stabilizes key structural motifs of σS2 to promote the assembly of σS-RNAP holoenzyme and also to facilitate formation of the RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

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Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

eLife ◽

10.7554/elife.50928 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 6

Author(s):

Juncao Xu ◽

Kaijie Cui ◽

Liqiang Shen ◽

Jing Shi ◽

Lingting Li ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Transcription Activation ◽

Initiation Factor ◽

Bacterial Cells ◽

E Coli ◽

Exchange Mass Spectrometry ◽

Transcription Initiation Factor ◽

Hydrogen Deuterium ◽

Promoter Dna

σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase–promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

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Activation of GCN2 by the ribosomal P-stalk

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813352116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4946-4954 ◽

Cited By ~ 43

Author(s):

Alison J. Inglis ◽

Glenn R. Masson ◽

Sichen Shao ◽

Olga Perisic ◽

Stephen H. McLaughlin ◽

...

Keyword(s):

Conformational Changes ◽

Principal Component ◽

Protein Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Hdx Ms ◽

Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

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