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 How brains surrender to sleep
23.06.2017 | IMP - Forschungsinstitut für Molekulare Pathologie GmbH

nachricht A new technique isolates neuronal activity during memory consolidation
22.06.2017 | Spanish National Research Council (CSIC)

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: Can we see monkeys from space? Emerging technologies to map biodiversity

An international team of scientists has proposed a new multi-disciplinary approach in which an array of new technologies will allow us to map biodiversity and the risks that wildlife is facing at the scale of whole landscapes. The findings are published in Nature Ecology and Evolution. This international research is led by the Kunming Institute of Zoology from China, University of East Anglia, University of Leicester and the Leibniz Institute for Zoo and Wildlife Research.

Using a combination of satellite and ground data, the team proposes that it is now possible to map biodiversity with an accuracy that has not been previously...

Im Focus: Climate satellite: Tracking methane with robust laser technology

Heatwaves in the Arctic, longer periods of vegetation in Europe, severe floods in West Africa – starting in 2021, scientists want to explore the emissions of the greenhouse gas methane with the German-French satellite MERLIN. This is made possible by a new robust laser system of the Fraunhofer Institute for Laser Technology ILT in Aachen, which achieves unprecedented measurement accuracy.

Methane is primarily the result of the decomposition of organic matter. The gas has a 25 times greater warming potential than carbon dioxide, but is not as...

Im Focus: How protons move through a fuel cell

Hydrogen is regarded as the energy source of the future: It is produced with solar power and can be used to generate heat and electricity in fuel cells. Empa researchers have now succeeded in decoding the movement of hydrogen ions in crystals – a key step towards more efficient energy conversion in the hydrogen industry of tomorrow.

As charge carriers, electrons and ions play the leading role in electrochemical energy storage devices and converters such as batteries and fuel cells. Proton...

Im Focus: A unique data centre for cosmological simulations

Scientists from the Excellence Cluster Universe at the Ludwig-Maximilians-Universität Munich have establised "Cosmowebportal", a unique data centre for cosmological simulations located at the Leibniz Supercomputing Centre (LRZ) of the Bavarian Academy of Sciences. The complete results of a series of large hydrodynamical cosmological simulations are available, with data volumes typically exceeding several hundred terabytes. Scientists worldwide can interactively explore these complex simulations via a web interface and directly access the results.

With current telescopes, scientists can observe our Universe’s galaxies and galaxy clusters and their distribution along an invisible cosmic web. From the...

Im Focus: Scientists develop molecular thermometer for contactless measurement using infrared light

Temperature measurements possible even on the smallest scale / Molecular ruby for use in material sciences, biology, and medicine

Chemists at Johannes Gutenberg University Mainz (JGU) in cooperation with researchers of the German Federal Institute for Materials Research and Testing (BAM)...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Plants are networkers

19.06.2017 | Event News

Digital Survival Training for Executives

13.06.2017 | Event News

Global Learning Council Summit 2017

13.06.2017 | Event News

 
Latest News

Quantum thermometer or optical refrigerator?

23.06.2017 | Physics and Astronomy

A 100-year-old physics problem has been solved at EPFL

23.06.2017 | Physics and Astronomy

Equipping form with function

23.06.2017 | Information Technology

VideoLinks
B2B-VideoLinks
More VideoLinks >>>