In order for
life to exist, inert genes must be translated into
action, action that occurs when proteins are made by the
ribosome. A large, complex machine, the ribosome is
poorly understood despite intensive experimental efforts
for decades. Key questions about the fundamental nature
of protein synthesis remain unanswered. For example, how
does the ribosome read the genetic code? And how do
certain antibiotics, so useful in reducing infections,
cripple ribosomes? In mammals, how are ribosomes
regulated, and how do certain viruses and cancers
circumvent this regulation? Probing the structure of
prokaryotic ribosomes provides the best approach for
understanding the highly-conserved functions of the
ribosome in all kingdoms of life. With ribosome
structures to 7-10 Å or higher resolution, we will be
able to compare the known biochemical, genetic, and
phylogenetic data to the medium-resolution electron
density map, revolutionizing the kinds of questions that
can be asked about the function of the ribosome during
the translation cycle. High-resolution ribosome
structures (3Å) will build the foundation for insight
into the "business end" of the genetic code
Crystallization
of 70S ribosome complexes: To investigate translational
fidelity, my structural studies will focus on 70S
ribosomal complexes. Transfer RNA is thought to bind to
the ribosome in at least seven different modes involving
both classical and hybrid states [1]. The classical
states and some of the hybrid states may form stable
ribosome complexes and produce well-ordered crystals.
Although the present crystals of 70S ribosomes are from a
prokaryotic thermophile, previous experiments on E. coli
ribosomes will guide our approaches to forming stable
complexes for crystallization. Other complexes that may
provide better crystals include those with elongation
factor G (EFG), EF-Tu/tRNA ternary complex bound to the
ribosome, or with antibiotics that target the ribosome.
In collaboration with Drs. Marat Yusupov, Gulnara
Yusupova, and Harry Noller, we have obtained crystals of
a 70S ribosome complex that diffract x-rays to a nominal
resolution of 5 Å. From these crystals, we have solved
the structure of the ribosome to 10 Å and are now
working to obtain phases for the structure factors to
about 7 Å. This is a comparable phasing resolution to
the initial nucleosome structure [2].
What will we be able to see at 10 Å or better
resolution? In the nucleosome core particle solved at 7
Å resolution, the DNA was clearly distinguishable from
proteins, as were the DNA major and minor grooves. In the
10 Å electron density maps of the ribosome, we are
beginning to see the major and minor grooves of RNA
double helices. We have also unambiguously identified the
positions of tRNA in the 3 of its binding states. Based
on the extensive biochemical data collected on similar
prokaryotic ribosomes, we have also begun to model the
RNA and protein positions in the 30S and 50S subunits.
While high-resolution structures of the ribosome will be
necessary to build complete models, our preliminary
modeling points to numerous biochemical experiments to
test ribosome function.
While the 70S electron density maps are still in the
process of refinement, we have taken the phases from one
of the heavy-atom derivatives to use in a control
experiment as a gauge of the quality of the phases. We
have collected 7-9 Å resolution data sets from two
different 70S ribosome complexes containing either an
intact tRNA or partial tRNA in the ribosomal P site. When
using the x-ray phases in combination with difference
amplitudes from these complexes ( |F|full tRNA
-|F|partial tRNA), at 10 Å resolution we obtain an
electron density map in which the location of the tRNA in
the P site is clearly seen. The major and minor grooves
in the RNA helices are clearly visible, and the position
of the 3'-CCA of the tRNA is prominent . From this
control experiment, we know that the heavy-atom phasing
is working very well so far.
The importance of cations in ribosome folding and
function: All of the fundamental activities of the
ribosome depend on ionic conditions. For example, as the
concentration of Mg2+ drops, 70S ribosomal
particles dissociate into 50S and 30S subunits. Further
lowering the Mg2+ concentration causes the
subunits to disassemble. In the other direction, ribosome
reconstitution has specific Mg2+ and polyamine
requirements [3]. Most importantly, translation fidelity
in vitro depends on a narrow range of ionic conditions,
above and below which ribosomes lose decoding accuracy.
These results, along with my discovery of a specific
magnesium ion core in the P4-P6 domain from the T.
thermophila group I intron [4], suggest there are
specific binding sites for Mg2+ in the
ribosome that play key roles in ribosome structure and
function.
We will use the 70S ribosome crystals to locate and
analyze these sites in a manner similar to the approach
used on the P4-P6 group I intron domain [4]. However, the
ribosome poses additional problems related to its size
and the resolution to which its structure is known. To
overcome the limits of resolution in a 7-10 Å map, I
will have to rely on markers like osmium (III) hexammine
and pentammine and lanthanides to identify potential
magnesium sites in the electron density [5]. In
combination with known biochemical data on important
phosphate oxygens and careful modeling of the RNA into
the electron density map, we will develop hypotheses as
to which parts of the ribosomal RNA bind metal ions.
The RNA we identify crystallographically in possible
magnesium binding sites will be tested for its functional
roles via phosphorothioate substitution interference [4].
Functional tests such as folding compactness or subunit
association will be used to separate active from inactive
RNAs, which can then be analyzed by iodine-EtOH cleavage
and reverse-transcription. Phosphorothioates which
interfere with Mg2+ binding should be
suppressed to some extent by manganese ion binding.
Combining the crystallographic data with tests of
ribosome activity may finally show the specific roles
metal ions play in translation.
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