Journal Article

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

Darst, Seth A, Lass-Napiorkowska, Agnieszka, Landick, Robert, Bae, Brian, Feklistov, Andrey

2015

Overview

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the −10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the −10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation. Inside cells, molecules of double-stranded DNA encode the instructions needed to make proteins. To make a protein, the two strands of DNA that make up a gene are separated and one strand acts as a template to make molecules of messenger ribonucleic acid (or mRNA for short). This process is called transcription. The mRNA is then used as a template to assemble the protein. An enzyme called RNA polymerase carries out transcription and is found in all cells ranging from bacteria to humans and other animals. Bacteria have the simplest form of RNA polymerase and provide an excellent system to study how it controls transcription. It is made up of several proteins that work together to make RNA using DNA as a template. However, it requires the help of another protein called sigma factor to direct it to regions of DNA called promoters, which are just before the start of the gene. When RNA polymerase and the sigma factor interact the resulting group of proteins is known as the RNA polymerase ‘holoenzyme’. Transcription takes place in several stages. To start with, the RNA polymerase holoenzyme locates and binds to promoter DNA. Next, it separates the two strands of DNA and exposes a portion of the template strand. At this point, the DNA and the holoenzyme are said to be in an ‘open promoter complex’ and the section of promoter DNA that is within it is known as a ‘transcription bubble’. However, it is not clear how RNA polymerase holoenzyme interacts with DNA in the open promoter complex. Bae, Feklistov et al. have now used X-ray crystallography to reveal the three-dimensional structure of the open promoter complex with an entire transcription bubble from a bacterium called Thermus aquaticus. The experiments show that there are several important interactions between RNA polymerase holoenzyme and promoter DNA. In particular, the sigma factor inserts into a region of the DNA at the start of the transcription bubble. This rearranges the DNA in a manner that allows the DNA to be exposed and contact the main part of the RNA polymerase. If the holoenyzyme fails to contact the DNA in this way, the holoenzyme does not bind properly to the promoter and transcription does not start. These findings build on previous work to provide a detailed structural framework for understanding how the RNA polymerase holoenzyme and DNA interact to form the open promoter complex. Another study by Bae et al.—which involved some of the same researchers as this study—reveals how another protein called CarD also binds to DNA at the start of the transcription bubble to stabilize the open promoter complex.

• Publisher: eLife Sciences Publications, Ltd,eLife Sciences Publications Ltd
• Subject: BASIC BIOLOGICAL SCIENCES / Biochemistry / Biophysics and Structural Biology / Crystallography, X-Ray / DNA, Bacterial - chemistry / DNA, Bacterial - metabolism / DNA-Directed RNA Polymerases - chemistry / DNA-Directed RNA Polymerases - metabolism / Holoenzymes - chemistry / Holoenzymes - metabolism / Models, Molecular / Nucleic Acid Conformation / open promoter compex / Promoter Regions, Genetic / Protein Binding / Protein Conformation / RNA polymerase / Thermus - chemistry / Thermus - enzymology / Thermus aquaticus / transcription / X-ray crystallography