A new super-sensitive microscope has been used to track the real-time motion of a
single protein. The microscope is able to zoom down to the scale of its individual atoms, revealing how genes are copied from DNA – a process essential to life.

The novel device allows users to achieve the highest-resolution
measurements ever, equivalent to the diameter of a single hydrogen
atom, says Steven Block, who designed it with colleagues at Stanford
University in California.

Block was able to use the microscope to track a molecule of DNA from an E.coli bacterium, settling a long-standing scientific debate about the precise method in which genetic material is copied for use.

The
molecular double-helix of DNA resembles a twisted ladder consisting of
two strands connected by “rungs” called bases. The bases, which are
known by the abbreviations A, T, G and C, encode genetic information,
and the sequence in which they appear “spell out” different genes.

Every
time a new protein is made, the genetic information for that protein
must first be transcribed from its DNA blueprint. The transcriber, an
enzyme called RNA polymerase (RNAP), latches on to the DNA ladder and
pulls a small section apart lengthwise. As it works its way down the
section of DNA, RNAP copies the sequence of bases and builds a
complementary strand of RNA – the first step in a new protein.

“For
years, people have known that RNA is made up one base at a time,” Block
says. “But that has left open the question of whether the RNAP enzyme
actually climbs up the DNA ladder one rung at a time, or does it move
instead in chunks – for example, does it add three bases, then jump
along and add another three bases.

In
order to settle the question, the researchers designed equipment that
was able to very accurately monitor the movements of a single DNA
molecule.

Block
chemically bonded one end of the DNA length to a glass bead. The bead
was just 1 micrometre across, a thousand times the length of the DNA
molecule and, crucially, a billion times its volume. He then bonded the
RNAP enzyme to another bead. Both beads were placed in a watery
substrate on a microscope slide.

Using
mirrors, he then focused two infrared laser beams down onto each bead.
Because the glass bead was in water, there was a refractive (optical
density) difference between the glass and water, which caused the laser
to bend and focus the light so that Block knew exactly where each bead
was.

But
in dealing with such small objects, he could not afford any of the
normal wobbles in the light that occur when the photons have to pass
through different densities of air at differing temperatures. So, he
encased the whole microscope in a box containing helium. Helium has a
very low refractive index so, even if temperature fluctuations
occurred, the effect would be too small to matter.

The
group then manipulated one of the glass beads until the RNAP latched on
to a rung on the DNA molecule. As the enzyme moved along the bases, it
tugged the glass bead it was bonded too, moving the two beads toward
each together. The RNAP jerked along the DNA, pausing between jerks to
churn out RNA transcribed bases. It was by precisely measuring the
lengths of the jerks that Block determined how many bases it
transcribed each time.

“The RNAP climbs the DNA ladder one base pair at a time – that is probably the right answer,” he says.

“It’s
a very neat system – amazing to be able see molecular details and work
out how DNA is transcribed for the first time,” said Justin Molloy, who
has pioneered similar work at the National Institute for Medical
Research, London. “It’s pretty incredible. You would never have
believed it could be possible 10 years ago.”

Steven Block at Stanford University.