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 Monitoring biodiversity with sound: how machines can enrich our knowledge
18.06.2019 | Georg-August-Universität Göttingen

nachricht Uncovering hidden protein structures
18.06.2019 | Universität Konstanz

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: The hidden structure of the periodic system

The well-known representation of chemical elements is just one example of how objects can be arranged and classified

The periodic table of elements that most chemistry books depict is only one special case. This tabular overview of the chemical elements, which goes back to...

Im Focus: MPSD team discovers light-induced ferroelectricity in strontium titanate

Light can be used not only to measure materials’ properties, but also to change them. Especially interesting are those cases in which the function of a material can be modified, such as its ability to conduct electricity or to store information in its magnetic state. A team led by Andrea Cavalleri from the Max Planck Institute for the Structure and Dynamics of Matter in Hamburg used terahertz frequency light pulses to transform a non-ferroelectric material into a ferroelectric one.

Ferroelectricity is a state in which the constituent lattice “looks” in one specific direction, forming a macroscopic electrical polarisation. The ability to...

Im Focus: Determining the Earth’s gravity field more accurately than ever before

Researchers at TU Graz calculate the most accurate gravity field determination of the Earth using 1.16 billion satellite measurements. This yields valuable knowledge for climate research.

The Earth’s gravity fluctuates from place to place. Geodesists use this phenomenon to observe geodynamic and climatological processes. Using...

Im Focus: Tube anemone has the largest animal mitochondrial genome ever sequenced

Discovery by Brazilian and US researchers could change the classification of two species, which appear more akin to jellyfish than was thought.

The tube anemone Isarachnanthus nocturnus is only 15 cm long but has the largest mitochondrial genome of any animal sequenced to date, with 80,923 base pairs....

Im Focus: Tiny light box opens new doors into the nanoworld

Researchers at Chalmers University of Technology, Sweden, have discovered a completely new way of capturing, amplifying and linking light to matter at the nanolevel. Using a tiny box, built from stacked atomically thin material, they have succeeded in creating a type of feedback loop in which light and matter become one. The discovery, which was recently published in Nature Nanotechnology, opens up new possibilities in the world of nanophotonics.

Photonics is concerned with various means of using light. Fibre-optic communication is an example of photonics, as is the technology behind photodetectors and...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

SEMANTiCS 2019 brings together industry leaders and data scientists in Karlsruhe

29.04.2019 | Event News

Revered mathematicians and computer scientists converge with 200 young researchers in Heidelberg!

17.04.2019 | Event News

First dust conference in the Central Asian part of the earth’s dust belt

15.04.2019 | Event News

 
Latest News

Uncovering hidden protein structures

18.06.2019 | Life Sciences

Monitoring biodiversity with sound: how machines can enrich our knowledge

18.06.2019 | Life Sciences

Schizophrenia: Adolescence is the game-changer

18.06.2019 | Life Sciences

VideoLinks
Science & Research
Overview of more VideoLinks >>>