February 28, 2013
Berkeley Lab Team Examines DNA To RNA Transcription
Alan McStravick for redOrbit.com — Your Universe Online
The Human Genome Project, a study intended to map out the structure of the 20,000-25,000 genes in human DNA, was begun in 1990 and concluded in 2003. The project, originally coordinated by the US Department of Energy and the National Institutes of Health, later partnered with the Wellcome Trust in the UK along with scientists from Japan, France, Germany and China. The act of mapping out human DNA and storing it in research databases was a huge leap forward in our understanding of a wide range of conditions, including many debilitating diseases.The next generation of research, building upon what was learned from the HGP, has culminated in a new study out of the Lawrence Berkeley National Laboratory (Berkeley Lab), which is run by the DOE. Their findings indicate a major advance in our understanding of just how genetic information is transcribed from DNA to RNA. The team developed a step-by-step look at the biomolecular machinery that reads the human genome.
“We´ve provided a series of snapshots that shows how the genome is read one gene at a time,” says biophysicist Eva Nogales, who led this research. “For the genetic code to be transcribed into messenger RNA, the DNA double helix has to be opened and the strand of gene sequences has to be properly positioned so that RNA polymerase, the enzyme that catalyzes transcription, knows where the gene starts. The electron microscopy images we produced show how this is done.”
Assisting with funding on this research is the NIH´s National Institute of General Medical Sciences. Paula Flicker of the NIH stated, “The process of transcription is essential to all living things so understanding how it initiates is enormously important. This work is a beautiful example of integrating multiple approaches to reveal the structure of a large molecular complex and provide insight into the molecular basis of a fundamental cellular process.”
Nogales, the corresponding author on the paper “Structural Visualization of Key Steps in Human Transcription Initiation” holds joint appointments with the Berkeley Lab, the University of California at Berkeley, and the Howard Hughes Medical Institute. Co-authors are Yuan He, Jie Fang and Dylan Taatjes. Their paper is to be published in the journal Nature.
In the field of molecular biology, the Holy Grail has been to try to understand the fundamental process of life wherein information contained in the genome of a living cell is used to generate biomolecules. It is these biomolecules that are responsible for the transmission of cellular activity. For those studying in the field, they hold the above to be their so-called “central dogma of molecular biology.” In this strong belief is held the idea that genetic information flows from DNA to RNA to proteins. It is this linear flow of information, initiated by an elaborate system of proteins operating in a highly choreographed method with stunning precision, which researchers want to understand more fully. It will only be with the achievement of an understanding of how this protein machinery works during transcription that scientists will be able to more accurately identify cellular malfunctions that could, for instance, turn cells cancerous or lead to a plethora of other problems.
The first step for Nogales and her research team involved the use of cryo-electron microscopy (cryo-EM). In cryo-EM, protein samples are flash-frozen at liquid nitrogen temperatures. This flash-freeze aids in the preservation of the structure of the protein allowing the team to carry out in vitro studies of the reconstituted and purified versions of the “transcription pre-initiation complex.” Located in this complex is a large assemblage of proteins comprised of RNA polymerase II (Pol II). Additionally, a class of proteins known as general transcription factors is contained here, as well. Each component in the complex works together seamlessly to ensure the accurate loading of DNA into Pol II at the beginning of the gene sequencing.
“There´s been a lack of structural information on how the transcription pre-initiation complex is assembled, but with cryo-EM and our in vitro reconstituted system we´ve been able to provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions during this step-by-step process,” Nogales says.
The key to the research model is the use of the in vitro reconstituted transcription pre-initiation complex. This method was developed by lead author and post-doctoral student Yuan He.
“This reconstituted system provided a model for the sequential assembly pathway of transcription initiation and was essential for us to get the most biochemically homogenous samples,” Nogales says. “Also essential was our ability to use automated data collection and processing so that we could generate all our structures in a robust manner.”
Perhaps the most interesting new detail yielded by He´s method of step-by-step cryo-EM imaging was how the transcription factor protein, known as TFIIF, actively engages the Pol II and promoter DNA to stabilize both a closed DNA pre-initiation complex as well as an open DNA-promoter complex. The research team also learned how the selection of a transcription start-site is regulated.
“Comparing the closed versus open DNA states led us to propose a model that describes how DNA is moved during the process of promoter opening,” says He. “Our studies provide insight into how THIIH uses ATP hydrolysis as a source of energy to actually open and push the DNA to the active site of Pol II.”
Nogales states she and her colleagues intend to further investigate the process associated with the DNA loading into Pol II. Additionally, they plan to include further transcription factors into the assembly that are required for the regulation of a gene´s expression.
“Our goal is to actually build a structural model of the entire — more than two million Daltons — protein machinery that recognizes and regulates all human DNA promoters,” Nogales says. “For now we have the structural framework that´s been needed to integrate biochemical and structural data into a unified mechanistic understanding of transcription initiation.”
This study, when viewed through the context of the scientific wilderness we were in just 23 short years ago before the launch of the HGP, is quite thrilling. The best and brightest minds are building off of the huge leap forward that was the HGP. It took only 10 years from its culmination to lead scientists and researchers to this significant point. The real wonder is, “What advances will we make in the next few years?”