Page 196 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology EN013D-617 July 27, 2001 11:42

236 Protein Synthesis

Only small stretches of rRNA are identical in the major- small subunit. Using similar methods several pairwise dis-
ity of organisms, and these are therefore predicted to be tances have been generated for the large subunit as well
critical for ribosomal function. but, with over twice as many proteins, the task is signifi-
More sequence conservation is observed for ribosomal cantly more difficult.
proteins than for rRNA. Structural similarities are signif- Together the accumulated biochemical and structural
icant enough that cross-species RNA interactions were data have generated several models of ribosome structure.
shown possible for proteins L1, L11, and S15. At a min- Several high-resolution structures of individual ribosomal
imum, then, the RNA-binding features of these proteins proteins or fragments of rRNA bound to proteins have also
are conserved. been determined. However, the piecewise reconstruction
of structural data seemed unlikely to provide an adequate
picture of the ribosome at the molecular level. Recently,
C. Structural Studies of the Ribosome
however, structures of isolated subunits and the assem-
The ribosome’s size challenged structural studies since its bled ribosome have been determined at a resolution that
first observation in electron micrographs nearly 50 years is already impressive and will continue to improve.
ago. With a molecular mass of 2500 kD and dimensions
˚
about 250 A on a side, the 70S ribosome is small for elec-
D. Recent High-Resolution Structures
tron microscopy but immense for structural studies (such
as NMR and X-ray crystallography) typically applied to Neither electron microscopy nor X-ray crystallography
single molecules. seemed suited to provide a high-resolution structure of the
The size and complexity of the ribosome have resulted ribosome, which was thought to be too small for the first
in the development and application of many different ex- and too large for the second method. Yet both have been
perimental approaches to probe the arrangement of pro- pushed to the limit to provide significant new structures
teins and rRNA. Electron microscopy hinted at the overall that are already explaining mechanistic features of protein
shape of individual subunits and the assembled ribosome. synthesis.
The delineation of these shapes has changed little even Cryoelectron microscopy (cryo-EM) has produced re-
with current high-resolution structures. The generation of markable images of the entire ribosome as a result of two
antibodies against ribosomal proteins, rRNA termini, and major advances in methodology—one experimental, one
modified bases allowed identification of their respective computational. Samples for cryo-EM (in this case of the
locations on the ribosome surface by means of immu- 70S E. coli ribosome) are frozen quickly, encased in vitre-
noelectron microscopy. Specific sites of protein–rRNA ous ice which lacks the crystals that can distort macro-
interactions were determined by chemical modification molecular structure. Following electron microscopy of
experiments, which generate a “footprint” of protein bind- these samples, a computer program digitally averages and
ing according to the differential reactivity of rRNA nu- aligns tens of thousands of images from single ribosomes
cleotides in the presence and absence of the protein. Simi- atdifferentangles.Theresultingreconstructionproducesa
larmodificationexperimentsalsoconfirmedthesecondary three-dimensional image at much higher resolution than
structure predictions made for rRNAs, using enzymes and any of the individual two-dimensional micrographs.
chemicals that react selectively with bases in either single- One of the first features observed by cryo-EM was the
stranded or helical regions. amount of visible deep clefts and “holes.” Intersubunit
More detailed information was obtained about the contacts are limited to a discrete number of “bridges” be-
orientation of proteins and rRNA, using reagents that gen- tween large and small subunits. These now appear to be
erate covalent cross-links between ribosomal neighbors. composed primarily of RNA. The relatively small inter-
Protein–protein, protein–RNA, and RNA–RNA cross- subunit surface is consistent with the need for subunits
links provided valuable constraints for model-building to dissociate following protein synthesis to accept a new
studies by limiting the distance that these components mRNA molecule.
could be placed from one another according to the length In addition to unoccupied 70S ribosomes, cryo-EM re-
of the cross-linker. A related approach has been the use of constructions are currently available for ribosomes con-
reagents that cleave rRNA. The cleavage patterns reveal taining various ligands. For example, tRNAs have been
parts of the rRNA that are either buried or exposed. observed in each of the three ribosomal binding sites
Neutron diffraction of ribosomal proteins has been used (A, P, and E), and ribosome-bound EF-Tu has also been
to estimate the arrangement of ribosomal components. By visualized. The variety of structures available at up to
˚
reconstituting pairs of deuterated proteins into the 30S 11 A resolution points out an advantage of cryo-EM over
subunit, distances between the protein pairs were gen- X-ray crystallography for ribosomal structure determina-
erated to produce a model of protein arrangement in the tion. Because sample preparation is rapid compared to