From DNA to RNA to protein

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From DNA to RNA to protein

http://www.news-medical.net/?id=18485

Of 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 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

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, a 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.

" a 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. "

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