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

Juncao Xu,Jing Shi,Yu Zhang,Yu Feng,Linlin You,Liqiang Shen,Bei Yang,Chengli Fang,Kaijie Cui,Lingting Li,Guoping Zhao

doi:10.7554/elife.50928

Abstract

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.

Highlights

Bacterial cells are capable of rapidly adapting to different ecological conditions through highly regulated dynamic switching among different gene-expression programs, and they do so by selectively activating distinct s-RNA polymerase (s-RNAP) holoenzymes (Osterberg et al, 2011)
The results suggest that Crl probably functions at the step of S-RNA polymerase–promoter DNA open complex (RPo) formation
The results show a weak but site-specific cleavage in the expected T-stretch site when the S-RNAP holoenzyme is present in the reaction (Figure 4D, lane 2); and again the presence of s

Summary

Introduction

Bacterial cells are capable of rapidly adapting to different ecological conditions through highly regulated dynamic switching among different gene-expression programs, and they do so by selectively activating distinct s-RNA polymerase (s-RNAP) holoenzymes (Osterberg et al, 2011). The alternative initiating factor, S ( known as 38 in Escherichia coli) is the master stress regulator that s s s protects many Gram-negative bacteria from detrimental environmental conditions (Lange and Hengge-Aronis, 1991). It plays indispensable roles in the biofilm formation, virulence, antibiotic tolerance, and antibiotic persistence of human pathogens including Salmonella enterica, Pseudomonas aeruginosa, and E. coli (Hansen et al, 2008; Murakami et al, 2005; Stewart et al, 2015; Wu et al, 2015).

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Conclusion