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When it comes to gene transcription, random pauses aren't quite so random, study finds
June 16, 2006Of the thousands of proteins produced in our cells, few are as important as the enzyme RNA polymerase (RNAP), which has the unique ability to faithfully copy genetic information from DNA. In fact, all organisms—from bacteria to people—depend on RNAP to initiate the complex process of protein synthesis. Despite its crucial role in cell biology, fundamental questions remain about how the RNAP enzyme actually works.
Now scientists from Stanford University and the University of Wisconsin-Madison have solved part of puzzle. Writing in the June 16 edition of the journal Cell, the research team found that a molecule of RNAP makes frequent pauses at specific sites along the DNA double helix. This finding comes on the heels of the team's 2003 discovery that RNAP enzymes routinely make thousands of brief stops ("ubiquitous pauses") when carrying out the vital task of transcribing genetic information from DNA to RNA—a process called transcription.
"Transcription of genes is terribly important," said study co-author Steven M. Block, professor of biological sciences and of applied physics at Stanford. "It's what determines the difference between the cells in your brain or your heart or your liver. All of your cells have exactly the same DNA, but what makes them different is that they transcribe different genes that code for different proteins."
From DNA to RNA to protein
Protein synthesis is strikingly similar in all organisms. It starts with DNA—the famous ladder-shaped double helix, whose rungs (or "bases") consist of four chemical units known by the abbreviations A, T, G and C. A typical DNA molecule contains thousands of genes that encode thousands of proteins, which are essential for life. Each gene consists of a set of DNA bases arranged in a unique sequence that carries explicit instructions for building a specific protein. But one misplaced letter in that sequence—a T substituted for a C, for example—could produce a damaged protein that causes a serious disease or birth defect.
Transcription, the first step in protein synthesis, begins when an RNAP enzyme unzips a small section of the DNA double helix where a gene is located. The enzyme then builds a new complementary strand of RNA by chemically copying ("transcribing") the gene one base at a time. RNAP will continue moving along the DNA strand until the entire gene sequence is transcribed onto the encoded RNA, which then serves as a template for building the actual protein.
Nanotechnology
To observe RNAP in action, Block and his colleagues use a custom-built "optical trap" housed in his Stanford lab. This sensitive instrument allows researchers to observe transcription in real time by trapping individual molecules of DNA and RNAP in minute beams of infrared light.
"Our measurements are accurate to one-tenth of a nanometer—the width of a single hydrogen atom," Block explained. "When you study an RNAP enzyme at that scale, you discover that it moves along the DNA for a while, and then for no apparent reason it appears to stop. Some pauses we've already figured out. The really long ones, which happen every 1,000 bases or so and last up to 30 minutes, often occur when the enzyme makes a mistake. Then, it's got to back up and correct the mistake. But for every one of those, there are roughly 10 ubiquitous pauses that only last about 1 second and occur every 100 bases or so—and their role is really something of a mystery."
Sequence dependent
To find the answer, Kristina M. Herbert, a graduate student in Block's lab and lead author of the Cell study, created experimental DNA templates using a special 240-base pair sequence that triggers one of two types of long pauses in RNAP—a "backtracking pause" associated with gene regulation in which the enzyme reverses direction briefly; or a "hairpin pause, " named for tiny hairpin-shaped structures that sometimes form when an RNA strand binds to itself.
"Kristina made these totally cool DNA templates that have the same 240-base pair sequence repeated over and over again eight times in a row," Block said. When molecules of RNAP were attached to the templates, they behaved as predicted, pausing briefly at all of the backtrack and hairpin pause sites, but not actually backtracking or forming hairpins.
"That's great," Block said. "It's telling us that the enzyme is doing just what it should. After all, it's seen the same sequence eight times in a row, so it had better do the same thing eight times in a row. It also paused at several other sites as well, which is interesting. Sometimes it paused longer, sometimes shorter, but the average was remarkably the same—about a second or so. We also discovered that it just didn't stop at any old sequence but at very specific places where there's a signal in the DNA that basically says, 'Pause here.'"
That signal, he added, occurred for specific sequences in the DNA "We found that there is always a G near a specific pause position, and always a T or a C at another nearby position," he said. "So the pause seems to be sequence dependent. It's not always the same duration every time, but it's more likely to pause at one of these sites than at any other sites in between, so it's not just some random phenomenon that happens every once in a while. If I'm running down the road and I trip, that would be a random phenomenon. But if I run down the road and every time I trip there's a pothole, then that's not random."
Some researchers have argued that all pauses might be associated with either hairpin formation or backtracking, but the Cell study contradicts that assumption. "Most ubiquitous pauses have nothing whatsoever to do with backtracking or hairpins," Block said. "We think ubiquitous pauses are the most common and probably most important kind of pause, and the models that some biochemists have been using are just wrong."
What causes pauses?
The study also addresses a long-standing question about enzyme memory: If an enzyme pauses at one DNA site, will it alter its behavior when it encounters the same sequence again? "It turns out that RNAP enzymes may have individual personalities, but no memories," Herbert explained. "That is, they exhibit a distinct behavior—they tend to pause more or less, but they don't seem to remember having paused."
Why does RNAP make these fitful stops and starts? "No one really knows what all these ubiquitous pauses are doing nor what really causes pauses. They may be there to act as some kind of a governor to control the speed of transcription," Block said. "The control of gene transcription is one of the most fundamental ways to regulate gene expression in general, and one way to control transcription is to control pausing. Cancer is an example of gene control gone amuck, so understanding the regulation of genes is critical to understanding cancer. Our results provide fresh insights into the control mechanisms that cells have for regulating the genes they express."
Stanford University
From DNA to RNA to protein
Protein synthesis is strikingly similar in all organisms. It starts with DNA—the famous ladder-shaped double helix, whose rungs (or "bases") consist of four chemical units known by the abbreviations A, T, G and C. A typical DNA molecule contains thousands of genes that encode thousands of proteins, which are essential for life. Each gene consists of a set of DNA bases arranged in a unique sequence that carries explicit instructions for building a specific protein. But one misplaced letter in that sequence—a T substituted for a C, for example—could produce a damaged protein that causes a serious disease or birth defect.
Transcription, the first step in protein synthesis, begins when an RNAP enzyme unzips a small section of the DNA double helix where a gene is located. The enzyme then builds a new complementary strand of RNA by chemically copying ("transcribing") the gene one base at a time. RNAP will continue moving along the DNA strand until the entire gene sequence is transcribed onto the encoded RNA, which then serves as a template for building the actual protein.
Nanotechnology
To observe RNAP in action, Block and his colleagues use a custom-built "optical trap" housed in his Stanford lab. This sensitive instrument allows researchers to observe transcription in real time by trapping individual molecules of DNA and RNAP in minute beams of infrared light.
"Our measurements are accurate to one-tenth of a nanometer—the width of a single hydrogen atom," Block explained. "When you study an RNAP enzyme at that scale, you discover that it moves along the DNA for a while, and then for no apparent reason it appears to stop. Some pauses we've already figured out. The really long ones, which happen every 1,000 bases or so and last up to 30 minutes, often occur when the enzyme makes a mistake. Then, it's got to back up and correct the mistake. But for every one of those, there are roughly 10 ubiquitous pauses that only last about 1 second and occur every 100 bases or so—and their role is really something of a mystery."
Sequence dependent
To find the answer, Kristina M. Herbert, a graduate student in Block's lab and lead author of the Cell study, created experimental DNA templates using a special 240-base pair sequence that triggers one of two types of long pauses in RNAP—a "backtracking pause" associated with gene regulation in which the enzyme reverses direction briefly; or a "hairpin pause, " named for tiny hairpin-shaped structures that sometimes form when an RNA strand binds to itself.
"Kristina made these totally cool DNA templates that have the same 240-base pair sequence repeated over and over again eight times in a row," Block said. When molecules of RNAP were attached to the templates, they behaved as predicted, pausing briefly at all of the backtrack and hairpin pause sites, but not actually backtracking or forming hairpins.
"That's great," Block said. "It's telling us that the enzyme is doing just what it should. After all, it's seen the same sequence eight times in a row, so it had better do the same thing eight times in a row. It also paused at several other sites as well, which is interesting. Sometimes it paused longer, sometimes shorter, but the average was remarkably the same—about a second or so. We also discovered that it just didn't stop at any old sequence but at very specific places where there's a signal in the DNA that basically says, 'Pause here.'"
That signal, he added, occurred for specific sequences in the DNA "We found that there is always a G near a specific pause position, and always a T or a C at another nearby position," he said. "So the pause seems to be sequence dependent. It's not always the same duration every time, but it's more likely to pause at one of these sites than at any other sites in between, so it's not just some random phenomenon that happens every once in a while. If I'm running down the road and I trip, that would be a random phenomenon. But if I run down the road and every time I trip there's a pothole, then that's not random."
Some researchers have argued that all pauses might be associated with either hairpin formation or backtracking, but the Cell study contradicts that assumption. "Most ubiquitous pauses have nothing whatsoever to do with backtracking or hairpins," Block said. "We think ubiquitous pauses are the most common and probably most important kind of pause, and the models that some biochemists have been using are just wrong."
What causes pauses?
The study also addresses a long-standing question about enzyme memory: If an enzyme pauses at one DNA site, will it alter its behavior when it encounters the same sequence again? "It turns out that RNAP enzymes may have individual personalities, but no memories," Herbert explained. "That is, they exhibit a distinct behavior—they tend to pause more or less, but they don't seem to remember having paused."
Why does RNAP make these fitful stops and starts? "No one really knows what all these ubiquitous pauses are doing nor what really causes pauses. They may be there to act as some kind of a governor to control the speed of transcription," Block said. "The control of gene transcription is one of the most fundamental ways to regulate gene expression in general, and one way to control transcription is to control pausing. Cancer is an example of gene control gone amuck, so understanding the regulation of genes is critical to understanding cancer. Our results provide fresh insights into the control mechanisms that cells have for regulating the genes they express."
Stanford University
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Gene Transcription: Mechanisms and Control by R. J. White (Author) Univ. of Glasgow, UK. Provides the non-expert with the established fundamentals of transcription, as well as some of the recent developments (up to January 2000). For researchers and postgraduate students. Two-tone illustrations. Softcover. |
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Immediate Early Genes and Inducible Transcription Factors in Mapping of the Central Nervous System Function and Dysfunction by Elsevier Science Hardbound. That molecular neurobiology has become a dominant part of neuroscience research can be credited to the discovery of inducible gene expression in the brain and spinal cord. This volume deals with genes, whose expression patterns in the vertebrate central nervous system were the first to be revealed and then the most extensively investigated over the last 15 years. Immediate early genes (IEG) and their protein products, especially those acting as regulators of transcription (inducible transcription factors, ITF) have proven to be very valuable tools in functional neuroanatomy and neurophysiology, as they are rapidly and transiently induced in specific neurons in response to various modes of stimulation. Thus, they have been used to map neuronal populations selectively responsive... | |
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The Gene Krupa Story, Vol. 4: Live Performances & Transcriptions by Gene Krupa | |
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Gene transcription in reproductive tissue: Karolinska Symposia on Research Methods in Reproductive Endocrinology. Transactions of the fifth symposium held ... 1972 (Acta endocrinologics. Supplementum) b | |
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Gene Structure and Transcription (In Focus) by Trevor Beebee (Author), Julian Burke (Author) Significant advances have been made in the study of gene structure and transcription since the publication of the first edition of this popular volume in 1988. The new edition updates all the sections where progress has been made and includes new figures and references. Substantial additions have especially been made to the sections on RNA processing and eukaryotic DNA-binding proteins - topics which have been the subject of intensive research in recent years. Like the first edition, this book will be invaluable to students in molecular biology, genetics, and biochemistry as it provides a succinct account of this important subject. | |
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Transcription by Taylor & Francis Knowledge of transcription has moved forward at a furious pace over recent years, and an understanding of the processes involved in gene regulation and expression has become an essential element in biochemistry, genome biology, molecular biology and molecular genetics. In this timely book, the authors present an accessible, yet comprehensive, coverage suitable for students at a senior undergraduate level, and for postgraduates needing an overview of the current state of play. Transcription shows examples of transcription systems for eukaryotes and prokaryotes, indicates methods for studying transcription, and surveys the whole topic of transcription from many perspectives. |
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Gene Transcription in Reproductive Tissue. by E. Diczfalusy (Author) | |
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Rb and Tumorigenesis (Medical Intelligence Unit) by Springer "Rb and Tumorigenesis" examines how recent advances have demonstrated the interaction of Rb with chromatin remodeling enzymes. This new title explores the potential roles of these interactions in Rb functions and provides some evidence that distinct Rb co-repressor may target different genes in different phases of the cell cycle. |
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5th Symposium: Gene Transcription in Reproductive Tissue, Stockholm 1972 by Diczfalusy (Author) | |
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