Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Nanotech instruments allow first direct observations of RNA ’proofreading’

26.11.2003


The RNA polymerase proofreading mechanism (Credit: E.. Abbondanzieri)


When Ralph Waldo Emerson said that nature pardons no mistakes, he wasn’t thinking about RNA polymerase (RNAP) - the versatile enzyme that copies genes from DNA onto strands of RNA, which then serve as templates for all of the proteins that make life possible.

Emerson’s comment notwithstanding, RNAP makes plenty of mistakes but also proofreads and corrects them before they have a chance to create abnormal proteins. The error-prone nature of RNAP is not surprising given the size of its task. In human cells, for example, the RNAP enzyme has to make precise genetic copies from a DNA double helix that consists of billions of chemical bases known as A, T, G and C. It works like this: After latching onto the double helix, RNAP pulls it apart and starts building new RNA molecules by copying one DNA base at a time.

With thousands of A’s, T’s, G’s and C’s to transcribe, RNAP sometimes gets confused and copies the wrong base. Such errors occur roughly once every 1,000 bases, but RNAP’s remarkable self-correcting mechanism manages to catch most of them.



’’If the error is allowed to propagate, it could result in a bad protein or a wrong protein, but RNAP is an incredibly smart enzyme,’’ says Steven M. Block, a professor of biological sciences and of applied physics at Stanford University. ’’When RNAP adds the wrong base, it backs up on the DNA helix a little bit, cleaves off the piece of RNA that has the bad base in it and starts up again. That’s the hypothesis, at least.’’

New experiments

In a new study in the journal Nature, Block and his colleagues present strong evidence to support this proofreading hypothesis. Their results - based on actual observations of individual molecules of RNAP - are posted on Nature’s website: http://www.nature.com. In another set of experiments published in the Nov. 14 issue of Cell magazine, the researchers discovered that RNAP makes thousands of brief pauses as it pries open and copies the DNA double helix.

’’Together these two papers push the study of single proteins to new limits,’’ Block said. ’’We’ve been able to achieve a resolution of three angstroms - the width of three hydrogen atoms - in our measurements of the progress of this enzyme along DNA. In so doing, we’ve been able to visualize a backtracking motion of just five bases that accompanies RNAP error-correction or proofreading.’’

Both studies were conducted using two-dimensional optical force clamps - unique instruments designed and built by the Block lab. Located in soundproofed and temperature-controlled rooms in the basement of Stanford’s Herrin labs, these devices allow researchers to trap a single molecule of RNAP in a beam of infrared light, and then watch in real time as it moves along a single molecule of DNA.

’’We’ve been able to reduce drift and noise in our instruments to such an extent that we can see the tiniest motions of these molecules, through distances that are less than their own diameters,’’ Block explained. ’’Studying one macromolecule at a time, you learn so much more about its properties, but these kinds of experiments were just pipedreams 15 years ago.’’

Stops and starts

In their experiments, the Block team conducted more than 300 observations of single RNAP molecules extracted from E. coli bacteria. Although structurally somewhat different from human RNAP, the E. coli enzyme plays a very similar role in the complex transfer from gene to RNA to protein.

Using the optical clamp, researchers found that RNAP does not move at a steady pace along the DNA double helix but rather undergoes a fitful series of unexplained starts and stops. ’’This enzyme is either full on or full off, as far as we can tell,’’ Block said. ’’It moves at about ten to 15 bases a second and pauses on average about once every 100 bases. Pausing is ubiquitous. About 97 percent are short pauses that last between 1.5 to 4.5 seconds. The other three percent are long pauses - from 20 seconds to over 30 minutes.’’

The study published in Cell focused on the short pauses, he added: ’’What we learned is that short pauses do not involve backtracking. What are these pauses? The answer is we don’t know.’’

The Nature study looked at the long pauses, which the researchers discovered only occur during backtracking - the hypothetical proofreading event during which RNAP is believed to slide backwards and snip off defective RNA. To test the hypothesis, the scientists added two proteins called GreA and GreB that are known to speed up the RNA cleaving process in E. coli. It turned out that adding GreA and GreB significantly shortened the backtracking pauses, providing strong evidence that backtracking and proofreading go hand in hand.

Medical consequences

Finding the answer could have significant ramifications for biology and for human health, observed physics graduate student Joshua W. Shaevitz, co-lead author of the Nature study.

’’When it comes to transcribing genetic code from DNA to RNA, fidelity is important,’’ he said. ’’If the protein comes out wrong, it may be lethal to the cell or to the organism.’’

Certain antibiotics are known to increase the error rate during translation from RNA to protein, added applied physics graduate student Elio A. Abbondanzieri, co-lead author of the Nature paper and co-author of the Cell study.

’’In the future we hope to study RNAP as it backs up in other situations when there is no mistake,’’ he said.

’’This research allows us to see a process essential to life at a level of detail never before possible,’’ said Catherine Lewis, chief of the biophysics branch in the National Institute of General Medical Sciences (NIGMS), which funded both studies. ’’It’s analogous to measuring the speed and direction of a single car, while other studies saw only the rush of traffic. This basic research will advance our understanding of how errors in transcription underlie disease and will pave the way for better tools to address such problems.’’

Other co-authors of the Cell study are Keir C. Neuman, a postdoctoral fellow in Stanford’s Department of Biological Sciences; Robert Landick of the University of Wisconsin; and Jeff Gelles of Brandeis University. Landick also co-authored the Nature study.


COMMENT: Steven M. Block, Departments of Biological Sciences and Applied Physics: 650-724-4046, sblock@stanford.edu

EDITORS: The study, ’’Backtracking by single RNA polymerase molecules observed at near-base-pair resolution,’’ is available on Nature magazine’s Website,http://www.nature.com. ’’Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking’’ is published in the Nov. 14 edition of the journal Cell. Additional information, videos and other images are available online at http://www.stanford.edu/group/blocklab/NatureBacktracking/.

Mark Shwartz | EurekAlert!
Further information:
http://www.stanford.edu/group/blocklab/
http://www.nigms.nih.gov/
http://www.nature.com

More articles from Life Sciences:

nachricht Nanoparticle Exposure Can Awaken Dormant Viruses in the Lungs
16.01.2017 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt

nachricht Cholera bacteria infect more effectively with a simple twist of shape
13.01.2017 | Princeton University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Designing Architecture with Solar Building Envelopes

Among the general public, solar thermal energy is currently associated with dark blue, rectangular collectors on building roofs. Technologies are needed for aesthetically high quality architecture which offer the architect more room for manoeuvre when it comes to low- and plus-energy buildings. With the “ArKol” project, researchers at Fraunhofer ISE together with partners are currently developing two façade collectors for solar thermal energy generation, which permit a high degree of design flexibility: a strip collector for opaque façade sections and a solar thermal blind for transparent sections. The current state of the two developments will be presented at the BAU 2017 trade fair.

As part of the “ArKol – development of architecturally highly integrated façade collectors with heat pipes” project, Fraunhofer ISE together with its partners...

Im Focus: How to inflate a hardened concrete shell with a weight of 80 t

At TU Wien, an alternative for resource intensive formwork for the construction of concrete domes was developed. It is now used in a test dome for the Austrian Federal Railways Infrastructure (ÖBB Infrastruktur).

Concrete shells are efficient structures, but not very resource efficient. The formwork for the construction of concrete domes alone requires a high amount of...

Im Focus: Bacterial Pac Man molecule snaps at sugar

Many pathogens use certain sugar compounds from their host to help conceal themselves against the immune system. Scientists at the University of Bonn have now, in cooperation with researchers at the University of York in the United Kingdom, analyzed the dynamics of a bacterial molecule that is involved in this process. They demonstrate that the protein grabs onto the sugar molecule with a Pac Man-like chewing motion and holds it until it can be used. Their results could help design therapeutics that could make the protein poorer at grabbing and holding and hence compromise the pathogen in the host. The study has now been published in “Biophysical Journal”.

The cells of the mouth, nose and intestinal mucosa produce large quantities of a chemical called sialic acid. Many bacteria possess a special transport system...

Im Focus: Newly proposed reference datasets improve weather satellite data quality

UMD, NOAA collaboration demonstrates suitability of in-orbit datasets for weather satellite calibration

"Traffic and weather, together on the hour!" blasts your local radio station, while your smartphone knows the weather halfway across the world. A network of...

Im Focus: Repairing defects in fiber-reinforced plastics more efficiently

Fiber-reinforced plastics (FRP) are frequently used in the aeronautic and automobile industry. However, the repair of workpieces made of these composite materials is often less profitable than exchanging the part. In order to increase the lifetime of FRP parts and to make them more eco-efficient, the Laser Zentrum Hannover e.V. (LZH) and the Apodius GmbH want to combine a new measuring device for fiber layer orientation with an innovative laser-based repair process.

Defects in FRP pieces may be production or operation-related. Whether or not repair is cost-effective depends on the geometry of the defective area, the tools...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

12V, 48V, high-voltage – trends in E/E automotive architecture

10.01.2017 | Event News

2nd Conference on Non-Textual Information on 10 and 11 May 2017 in Hannover

09.01.2017 | Event News

Nothing will happen without batteries making it happen!

05.01.2017 | Event News

 
Latest News

Energy-Efficient Building Operation: Monitoring Platform MONDAS Identifies Energy-Saving Potential

16.01.2017 | Trade Fair News

Designing Architecture with Solar Building Envelopes

16.01.2017 | Architecture and Construction

Sensory Stimuli Control Dopamine in the Brain

13.01.2017 | Life Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>