Corporate Banner
Satellite Banner
Proteomics
Scientific Community
 
Become a Member | Sign in
Home>News>This Article
  News
Return

A New View of Transcription Initiation

Published: Monday, March 04, 2013
Last Updated: Monday, March 04, 2013
Bookmark and Share
Reading the human genome.

The human genome is contained within a vast jumble of DNA. Its 20,000 or so genes are concealed within strings of As, Ts, Gs, and Cs, and each gene must be turned on at the right time and in the right cells.

For the first time, scientists have glimpsed the cellular machinery that accomplishes that feat, as it assembles directly on the DNA and readies it for transcription into RNA, the first step in protein production.

“We’ve described the assembly of the machinery that allows the human genome to be read one gene at a time. This molecular step is critical is transforming DNA ultimately into the protein repertoire that carries out all the functions of a cell,” says Eva Nogales, the Howard Hughes Medical Institute investigator who led the research. Nogales and her team published their work online in the journal Nature on February 27, 2013.

The enzyme that carries out transcription, RNA polymerase II, is very efficient at copying the information encoded in DNA into an RNA molecule. “But the polymerase is completely incapable of detecting the beginning of a gene,” says Nogales, whose lab is at the University of California, Berkeley.

Likewise, the polymerase cannot do its work until the two strands of the DNA double helix have been separated to reveal their sequence. To accomplish these tasks, a bulky complex of proteins called the pre-initiation complex is assembled each time transcription begins.

Together, the proteins in the pre-initiation complex find a gene’s start site, prepare the DNA, and set the polymerase on its way.

“This machinery has to find the beginning of the gene in the huge ocean of DNA that makes up the genome,” explains Nogales. Once there, it opens the double helix and positions the polymerase in exactly the right place, ensuring that transcription begins with the correct letter in the DNA code.

Researchers knew that it takes a core complex of at least six different factors-TATA-binding protein (TBP), TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH-to initiate transcription of human genes.

Based on biochemical experiments, they knew the order in which these transcription factors arrived at the start site and joined the complex, and they had an idea of each one’s contributions to transcription initiation. But no one had actually visualized the molecules in action, so the molecular details of how they functioned remained unknown.

The main obstacle to visualizing the pre-initiation complex, Nogales says, was obtaining enough of each of its components for structural studies. Several of the components in the complex are not amenable to the techniques scientists often rely on to produce large quantities of the protein in the lab.

Instead, Nogales and her colleagues isolate those proteins directly from human cells. Because the transcription factors are not abundant inside cells, the quantities that can be purified are small.

“Things like x-ray crystallography and NMR”-common techniques for structural studies that require large amounts of protein-“were completely out of the question,” Nogales says. “That’s why very few structures of any of these intermediates have been obtained.”

Instead, they turned to a technique called cryo-electron microscopy (cryo-EM), in which samples are flash frozen and then viewed through an electron microscope. Because the proteins do not have to be crystallized, researchers see them in their native state. Further, Nogales explains, “we need very little volume at very low concentrations to do cryo-EM.”

Nogales and her team wanted to watch as the pre-initiation complex assembled at a gene’s start site, so they created a series of cryo-EM snapshots. In a test tube, they allowed a minimal form of the complex to self assemble on a strand of DNA: a cluster including TBP, TFIIA, TFIIB, and the RNA polymerase II.

They froze the complex in this state and captured images to generate a 3D structure. All the pieces in this simple form of the complex were known, so Nogales says this allowed them to confirm that their technique was consistent with previous crystallographic images. They then added TFIIF, TFIIE, and TFIIH one by one, capturing three more snapshots.

In cells, the pre-initiation complex remains on the DNA until the polymerase begins transcription and physically moves away from the start site. Before this can happen, the pre-initiation complex must use energy to open the double helix and push the DNA into the pocket of the polymerase where transcription occurs.

Nogales and her team mimicked this state in the test tube by altering the sequence of DNA on which the transcription factors and polymerase assembled, adding a short segment of RNA. “It is the equivalent of the polymerase having engaged the open DNA and started to add a few nucleotides of RNA,” Nogales explains. Again, they froze the complex and captured images with cryo-EM.

The stop-motion movie they have created from their snapshots reveals several key features. It shows, for example, how TFIIF stabilizes the complex by engaging both the polymerase and the DNA.

TFIIF was known to be important for lining the polymerase up at a gene’s start site, and the new images show how it allows the polymerase to bind to the DNA double helix, then nudges the polymerase to just the right position on the DNA.

“This initial engagement sets things in the right position, so that when TFIIH pushes on the DNA, it will be moved into the right place,” Nogales explains. It also reveals how the same factor ensures that the double helix will unwind and separate at the right place, where part of TFIIF physically inserts itself between the strands to prevent them from coming back together.

“We have seen the molecular details of how all the proteins come together and interact with each other and with the DNA, thus gaining insight into how the cell determines at which point of the DNA to start transcribing into RNA, and actually how the DNA is opened and inserted into the active site in the polymerase,” Nogales says. “But this complex is really only the beginning of the story.”

“If we want to get at what is different between one gene and another, we have to start building up even larger complexes,” she says. So far, her team has recreated the formation of the most fundamental portion of the pre-initiation complex-the part that assembles every time any human gene is transcribed. But in living cells, these proteins never act alone. The next step, Nogales says, will be to begin to add in the factors that allow the transcription machinery to recognize genes in a regulated fashion. “As genomes get bigger, their regulatory systems get more complex,” Nogales says.

That complexity, she says, enables cells to fine tune gene expression-and understanding it is essential for understanding the complexity arising from the human genome.


Further Information
Access to this exclusive content is for Technology Networks Premium members only.

Join Technology Networks Premium for free access to:

  • Exclusive articles
  • Presentations from international conferences
  • Over 2,500+ scientific posters on ePosters
  • More than 3,700+ scientific videos on LabTube
  • 35 community eNewsletters


Sign In



Forgotten your details? Click Here
If you are not a member you can join here

*Please note: By logging into TechnologyNetworks.com you agree to accept the use of cookies. To find out more about the cookies we use and how to delete them, see our privacy policy.

Related Content

Spontaneous Mutations Play a Key Role in Congenital Heart Disease
New research shows that about 10 percent of these defects are caused by genetic mutations that are absent in the parents of affected children.
Monday, May 13, 2013
Search for Epigenetic Decoder in Brain Cells Leads Scientists to Rett Syndrome
New analysis suggests that MeCP2 recognizes 5hmC in the brain and facilitates activation of the genes.
Monday, December 31, 2012
HHMI’s Robert Lefkowitz Awarded 2012 Nobel Prize in Chemistry
Robert Lefkowitz and Brian K. Kobilka are the recipients of the 2012 Nobel Prize in Chemistry for studies of G-protein coupled receptors.
Thursday, October 11, 2012
Autism Gene Screen Highlights Protein Network for Howard Hughes Medical Institute Scientists
Over the past decade, scientists have added many gene mutations to the list of potential risk factors for autism spectrum disorders -- but researchers still lack a definitive explanation of autism’s cause.
Thursday, April 05, 2012
Protein-Folding Game Taps Power of Worldwide Audience to Solve Difficult Puzzles
Extended efforts could pay off in the design of new proteins that help fight disease, sequester carbon, or clean up the environment.
Monday, August 09, 2010
Mutations in Different Cells Cooperate to Set the Stage for Cancer
HHMI researchers have shown that distinct cancer-causing mutations in neighboring cells can cooperate to produce tumors.
Friday, January 15, 2010
Scientists Identify New Genetic Culprit for Intellectual Disability
HHMI researchers identified a genetic mutation that plays a role in intellectual disability.
Monday, December 14, 2009
Sticklebacks Hone Defenses through Small DNA Deletions
A single genetic adjustment is enough to help a small fish make a big change, HHMI researchers find.
Friday, December 11, 2009
Studies Begin to Shape New Image of DNA
Researchers to develop a new picture of DNA that shows the molecule’s more dynamic side, which is capable of morphing into a large number of complex shapes.
Monday, November 09, 2009
Diagnosis Emerges from Complete Sequencing of Patient's Genes
HHMI researchers have identified a gene mutation that was responsible for the patient’s disease, but had not been suspected based on clinical observations.
Wednesday, October 28, 2009
Diagnosis Emerges from Complete Sequencing of Patient's Genes
Howard researchers used high-throughput DNA sequencing technology to identify a gene mutation that was responsible for the patient’s disease.
Tuesday, October 20, 2009
A Proliferation of Amyloid Arrangements
New research shows that variations in each fibril-forming protein’s arrangements may represent a protein-based system of inheritance between cells that parallels the genetic code.
Tuesday, August 25, 2009
Study Pinpoints Genetic Drivers of Lung Cancer’s Spread
Howard Hughes Medical Institute investigator find that lung cancer uses to seed deadly new tumors in the brain, bone marrow, and other organs.
Friday, July 03, 2009
New Strategy Reveals Targets for MicroRNA Gene Regulation
Researchers use HITS-CLIP technique to map the binding points of scores of different microRNAs throughout a genome in living mouse or human tissue.
Friday, June 19, 2009
New Studies Reveal Broad, Hidden Network that Lets Tumors Thrive
Howard researchers identify potential new drug targets for cancers long deemed “untouchable” due to the type of genetic mutation they contain.
Monday, June 01, 2009
Scientific News
Liquid Biopsies: Utilization of Circulating Biomarkers for Minimally Invasive Diagnostics Development
Market Trends in Biofluid-based Liquid Biopsies: Deploying Circulating Biomarkers in the Clinic. Enal Razvi, Ph.D., Managing Director, Select Biosciences, Inc.
Self-Assembling, Biomimetic Membranes May Aid Water Filtration
A synthetic membrane that self assembles and is easily produced may lead to better gas separation, water purification, drug delivery and DNA recognition, according to an international team of researchers.
Crystal Clear Images Uncover Secrets of Hormone Receptors
NIH researchers gain better understanding of how neuropeptide hormones trigger chemical reactions in cells.
Error Correction Mechanism in Cell Division
Cell biologists have reported an advance in understanding the workings of an error correction mechanism that helps cells detect and correct mistakes in cell division early enough to prevent chromosome mis-segregation and aneuploidy, that is, having too many or too few chromosomes.
Crucial for Stem Cell Survival Protein Identified Using Editing Tool CRISPR
A team of University of Wisconsin-Madison engineers has identified a protein that is integral to the survival and self-renewal processes of human pluripotent stem cells (hPSC).
Sorting Through Cellular Statistics
Aaron Dinner, professor in chemistry, and his graduate student Herman Gudjonson are trying to read the manual of life, DNA, as part of the Dinner group’s research into bioinformatics—the application of statistics to biological research.
First Artificial Ribosome Designed
Researchers at the University of Illinois at Chicago and Northwestern University have engineered a tethered ribosome that works nearly as well as the authentic cellular component, or organelle, that produces all the proteins and enzymes within the cell.
The Genetic Roots of Adolescent Scoliosis
Scientists at the RIKEN Center for Integrative Medical Sciences in collaboration with Keio University in Japan have discovered a gene that is linked to susceptibility of Scoliosis.
HIV Susceptibility Linked to Little-Understood Immune Cell Class
High levels of diversity among immune cells called natural killer cells may strongly predispose people to infection by HIV, and may be driven by prior viral exposures, according to a new study.
New Tech Enables Epigenomic Analysis with a Mere 100 Cells
A new technology that will dramatically enhance investigations of epigenomes, the machinery that turns on and off genes and a very prominent field of study in diseases such as stem cell differentiation, inflammation and cancer has been developed by researchers at Virginia Tech.
Scroll Up
Scroll Down
Skyscraper Banner

Skyscraper Banner
Go to LabTube
Go to eposters
 
Access to the latest scientific news
Exclusive articles
Upload and share your posters on ePosters
Latest presentations and webinars
View a library of 1,800+ scientific and medical posters
2,500+ scientific and medical posters
A library of 2,500+ scientific videos on LabTube
3,700+ scientific videos
Close
Premium CrownJOIN TECHNOLOGY NETWORKS PREMIUM FREE!