Timing is everything, it seems, even in science. A
team led by Johns Hopkins scientists has unraveled the
first step in translating genetic information in order to
build a protein, only to find that it's not one step but
In a series of experiments, the scientists found that
when yeast's protein-building machinery recognizes the
starting line for a gene's instructions, it first alters
its structure and then releases a factor known as eIF1, a
step necessary to let it continue reading the assembly
instructions. Even though yeast are the most primitive
relatives of humans, the protein-building machinery, or
ribosomes, of each are quite similar.
"The idea is to really know at the molecular level how
life is put together," said Jon Lorsch, professor of
biophysical chemistry, one of the departments in Johns
Hopkins' Institute for Basic Biomedical Sciences. "We see
disease largely as an incorrect timing event — the
wrong thing happening at the wrong time, or the lack of the
As a result, Lorsch studies the timing of how the
ribosome complex itself assembles and how other factors
come and go as it translates genetic information to build
proteins, the workhorses of cells. If the ribosome doesn't
start in the right place along a gene's instructions, it
will make the wrong protein, which can kill the cell or
lead to disease.
"The ribosome is the end stage of gene expression, and
gene expression keeps us alive and causes disease," Lorsch
said. "If we can better understand how the ribosome works,
perhaps we can harness it to help us fix disease."
Already, scientists knew that without eIF1, the
ribosome can start reading the gene's RNA instructions at
places other than a particular three-block piece of RNA
known as the "start codon." And excessive amounts of eIF1
are associated with cardiac hypertrophy, or an enlarged
While eIF1's role in cardiac hypertrophy remains a
mystery, the new discovery reveals exactly how eIF1
regulates the ribosome's activity. The research team has
demonstrated that eIF1's mere presence on the yeast
ribosome prevents the machinery from getting started. Only
after its release from the complex can the ribosome start
"No one had any idea when eIF1 was released from the
ribosome, or that its release might serve an important
purpose, so this was a completely unexpected result," said
graduate student David Maag, first author of the paper.
"It's impossible to know for sure whether eIF1 is
released completely in living creatures, but in our
laboratory experiments, that is clearly the case," Lorsch
added. "Even if it isn't released completely in intact
cells, our results would indicate that it must be very
loosely associated for translation [protein building] to
To monitor what was happening to eIF1, the researchers
tagged it and a related part of the ribosome with different
fluorescent chemicals. When two fluorescently labeled
molecules are near one another, the fluorescent chemicals
subtly interact, which changes the color or wavelength of
light that is given off. If the distance between the
fluorescent molecules changes, the color of the emitted
light changes as well.
The researchers successfully used this phenomenon,
known as fluorescence resonance energy transfer, or FRET,
to monitor the relationship between eIF1 and its relative
as the ribosome complex assembled and after RNA was added
to the mix.
"We weren't even sure the two fluorescent molecules
would be close enough together to create a FRET signal at
all," Maag said. "We were very pleased just to be able to
monitor it, and then we were surprised and pleased by what
we saw next."
They had expected--or at least hoped--to see a shift
in the color of light once the RNA was mixed in. Instead,
they saw two shifts in the color given off. First, there
was a slight shift, indicating a small change in the
distance between eIF1 and its relative, and then a much
larger shift, indicating a much bigger separation.
To prove eIF1 was being released from the ribosome
complex, the researchers examined how fast the pieces of
the ribosome come together, and how long it takes them to
fall apart under various circumstances. Their results
support the idea that two separate steps take place once
the instruction's starting point is found: first a
structural change in the ribosome complex, and then release
Lorsch's goal is to know the five "Ws" and one "H"
that affect timing of all the ribosome's pieces and
activities, but unraveling every what, when, where, why,
who and how is no small task — roughly 27 bits like
eIF1 play a role at one point or another. To tackle the
problem, Lorsch and his colleagues move between "timing"
studies of the ribosome's molecular comings and goings, and
genetic studies that create mutant ribosome parts, which
likely affect ribosome function — and change its
The study appears in the Jan. 8 issue of Molecular
Cell. The authors are Maag and Lorsch, of Johns Hopkins;
Christie Fekete, of the National Institute of Child Health
and Human Development; and Zygmunt Gryczynski, of the
University of Maryland School of Medicine.
The Johns Hopkins researchers were funded by the
National Institute of General Medical Sciences and the
American Heart Association.