Visualization of two architectures in class-II CAP-dependent transcription activation.

Wei Shi,Bin Liu,Zigang Dong,Yibin Deng,Yanan Jiang

doi:10.1371/journal.pbio.3000706

Abstract

Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo–electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism.

Highlights

Transcription, the first step of gene expression, is regulated by various transcription factors
The carboxyl-terminal domain of the alpha subunit (αCTD) of RNA polymerase (RNAP) is invisible in density maps, a specific DNA sequence for its binding is included in the DNA scaffold
In the state 1 structure, further local resolution map showed the almost invisible density on the DNA portion for αCTD binding, the presence of gaps in the DNA part that is next to the αCTD binding portion, and the relatively weak density in the parts of cAMP receptor protein (CAP) dimer close to these gaps and the β flap tip (β-FT)

Summary

Introduction

Transcription, the first step of gene expression, is regulated by various transcription factors. How these transcription factors modulate RNA polymerase (RNAP) is significant for understanding the mechanisms of transcription and gene regulation. Cyclic AMP (cAMP) receptor protein (CAP) is a classic dimeric global transcription factor, which is activated by its allosteric effector cAMP [1,2,3]. CAP activates and initiates transcription on more than 100 promoters in E. coli [1], mainly via two different mechanisms: class-I and class-II, according to its interaction mode with RNAP holoenzyme [4,5,6], the main enzyme comprising a five-subunit core enzyme (α2ββ0ω) and a sigma factor, which is responsible for RNA synthesis in cells [7,8]. On class-I promoters, such as lac promoter, CAP binds at the −61.5 site of the promoter and interacts with RNAP holoenzyme by the carboxyl-terminal domain of the alpha

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Conclusion