When an enzyme takes a break

Access and retrieval of the genetic information stored in DNA is a fundamental process that requires tight regulation. Research by HFSP fellow Albert Weixlbaumer of The Rockefeller University has shed light on the molecular mechanism underlying a specific type of regulation, called transcriptional pausing, revealing more information about the first step of gene expression.

HFSP Long-Term Fellow Albert Weixlbaumer and colleagues
authored on Mon, 18 March 2013

In order to convert the information encoded in our DNA (our genotype) to who we are and what we look like (our phenotype), every living cell needs to retrieve the genetic information stored in DNA. The first step in this information retrieval, where DNA is copied into RNA, is called transcription. The central player carrying out transcription is a multi-subunit protein enzyme called DNA dependent RNA polymerase. RNA polymerase comprises a universally conserved core architecture meaning the basic mechanisms underlying transcription are the same from the simplest bacterial cell all the way to humans. Because of its fundamental role, transcription affects every aspect of biology and a wide variety of regulatory processes target RNA polymerase. The advents of high-resolution crystal structures of RNAPs from bacteria and yeast have revolutionized our understanding of transcription because we are now beginning to comprehend molecular level events in a better way. Significant progress has been made based on several crystal structures of proteins involved in initiation, the first phase of transcription, where RNA polymerase localizes the transcription start site and initiates DNA-template directed RNA synthesis. Elongation of transcription, the second phase, during which rapid RNA synthesis takes place, is also well understood. Termination on the other hand, the third and final phase of transcription, is not well understood on a mechanistic or structural level.

 

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Figure: (A) The clamp domain of RNA polymerase (green) opens as a result of entering the paused state. (B) A schematic representation of the model for entering the elemental paused state. During translocation (movement of RNA polymerase along the DNA template), the enzyme has a chance to relax, and enter the elemental paused state. This state is characterized by a fully open clamp domain, a kinked Bridge Helix blocking the active site of the enzyme and a widened RNA exit channel.

One way to control and regulate gene expression is through a process called transcriptional pausing, in which a transcribing RNA polymerase transiently adopts a catalytically inactive state during the elongation cycle. Pausing plays a paramount role in the regulation of gene expression in all kingdoms of life. In bacteria, RNA polymerase pauses to allow i) coordination of transcription and protein synthesis, ii) correct folding (i.e. adoption of the correct 3-dimensional structure) of nascent RNAs, iii) time for regulatory factors to bind the transcribing complex, and iv) termination of transcription. Because the paused RNAP plays a role in regulation as well as termination of transcription it is a particularly interesting state, which lacked mechanistic understanding.

Work by Albert Weixlbaumer, a postdoc with biophysicist Seth A. Darst at The Rockefeller University, in collaboration with biochemist Robert Landick, has now shed light on the mechanism of transcriptional pausing and provides a framework for understanding transcriptional termination. Paused RNA polymerase elongation complexes were formed and biochemically characterized in vitro. These artificially formed complexes exhibit the same kinetic behavior as in vivo and suitable specimens were chosen as targets for structural studies by X-ray crystallography to give a detailed, 3-dimensional picture at the atomic level.

Three crystal structures of RNAP from two different species in a state of transcriptional pausing gave a detailed picture of this process. The structures, combined with the biochemical and kinetic results, provide mechanistic insights into an important question in biology. The paused RNAP relaxes and adopts a different, low-energy conformation compared to the active, transcribing complex, resulting in a number of critical consequences: i) the clamp, a mobile domain of RNA polymerase, opens resulting in loosening of the contacts to the bound DNA and RNA; ii) the bridge helix, an element of RNA polymerase, kinks and directly blocks the active site of the enzyme explaining how catalysis is inhibited; iii) the conformation of the RNA/DNA hybrid is altered, suggesting the complex is primed for termination; and iv) the RNA exit channel, a narrow protein channel through which the nascent RNA threads through, becomes wider as a result of pausing. The latter conformational change provides space for an RNA hairpin structure to form in the otherwise too narrow exit channel, explaining how a hairpin stabilizes the paused state by serving as a steric wedge. In addition, intrinsic transcription terminators also form an RNA hairpin structure in the nascent transcript and the same rational explains why a paused state must be an obligatory termination intermediate.

Reference

Structural Basis of Transcriptional Pausing in Bacteria.  Albert Weixlbaumer, A., Katherine Leon, K., Robert Landick, and Seth A. Darst (2013). Cell, 152(3), 431–441. doi:10.1016/j.cell.2012.12.020

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