Microscopy of Ribosome Structure and Function

1
Microscopy of Ribosome Structure and Function
Justin Levy, Roger Ndindjock, James Potter,
Justin Quon, Sam Veihmeyer
Current Mechanism of Translation
Early Microscopy In the late 50s, scientists
began seriously considering the structure of the
ribosome. Light microscopes had reached their
theoretical limit twenty years prior at
magnifications of 500X or 1000X. 10,000X
magnification was needed to view organelle
structures and much higher magnification was
needed to visualize protein structure. The need
for more advanced microscopy techniques was
apparent.
Neutron Diffraction Neutron Diffraction (ND) was
developed in 1946 by Clifford G. Shull. In ND,
neutrons are accelerated in a magnetic field and
fired at the sample. Diffraction occurs when
neutron waves (approximately 0.1 nm) encounter
obstacles that are similar in size. The beam of
neutrons is not affected by atomic electron
clouds and will be diffracted only by atomic
nuclei. In this regard, ND is superior to EM or
X-Ray crystallography whose probes are affected
by atomic electron density. Small-Angle-Neutron-S
cattering (SANS) is a type of ND used for
determining the structures of protein that relies
on neutron scattering for detection. This
technique was vital to the determination of tRNA
locations in the mechanism for translation. SANS
was also used to determine the ribosomes atomic
structure.
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1 Initiation factor binding
2 Binding of tRNA with associated factors to
form preinitiation complex
2
Electron Microscopy Since its initial
development by Max Knoll and Ernst Ruska in 1931,
electron microscopy (EM) has been of pivotal
importance to the study of the ribosome. In EM,
a beam of highly energetic electrons is directed
at the sample to provide up to 500,000X
magnification. Invaluable topological,
morphological, compositional, and
crystallographic insight can be obtained.
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3 eIF4G/eIF4A binding to preinitiation complex
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4 Association of preinitiation factor with mRNA
Eukaryotic 80S Ribosome
Initiation
40S Subunit
60S Subunit
5 Small ribosome subunit scans for start codon
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28S rRNA
5.8S rRNA
18S rRNA
5S rRNA
49 Ribosomal Proteins
33 Ribosomal Proteins
6 Associated proteins falls off small subunit,
large subunit attaches and translation begins
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Large Subunit Atomic Structure (From
http//en.wikipedia.org/wiki/ImageRibosome_50s.pn
g)
(From http//www.emunix.emich.edu/rwinning/geneti
cs/pics/transl1.jpg)
Common Types of Electron Microscopes 1.
Tunneling EM Electrons pass through sample 2.
Scanning EM After intial bombardment, secondary
electrons leave the sample to be detected. 3.
Reflection EM After incidence, reflected
electrons are detected
X-Ray Crystallography Australian William Henry
Bragg and his son William Lawrence Bragghas won
the 1915 Noble Prize in Physics for the invention
of the X-Ray Crystallography (XRC) method. Due
to difficulties in crystallization, ribosomes
were not analyzed using XRC until the
illuminating work by Yonath and Wittman in 1980.
The 50S bacterial ribosome crystals prepared by
Yonath and Wittman diffracted X-rays to 3.5 Å and
provided scientists with a great deal of
structural data. The structure of the 30S
subunit was later revealed. In XRC,
crystallized sample deflects X-rays to produce a
diffraction pattern. This pattern corresponds to
the number of atoms in the molecule. Heavier
atoms scatter X-rays more effectively.
Diffraction patterns are visualized from several
angles to provide organizational information
about the molecular components.
Elongation
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1 Charged tRNAs (aminoacyl-tRNAs) bind to
elongation factor eEF-1 in the presence of GTP
2
2 The complex enters the empty A-site on a
ribosome carrying an initiator Met-tRNA
Cryo-Electron Microscopy Cryo-Electron
Microscopy (CEM) is an EM technique that involves
freezing biological samples in order to preserve
the hydrated state of the specimen. This also
provides protection in the high-radiation, low
pressure environment during observation. Through
CEM, binding sites of tRNA and elongation factors
were elucidated. CEM also helped distinguish
between the subunits of the ribosome. A key
consideration in CEM is the formation of vitreous
ice. Liquid ethane is used (-185C) instead of
liquid nitrogen (-195C) because of increased
heat capacity N2(l) will boil off in the process
of freezing, permitting unwanted ice crystals to
form.
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3 After the correct codon-tRNA binding, the
growing polypeptide in the P site is transferred
to the A site. Peptidyl transferase attaches
the amino acid to the nascent peptide chain
4
References Ban, Nissen, et al. The Complete
Atomic Structure of the Large Ribosomal Subunit
at 2.4 Angstrom Resolution. Science. 289.
2000. Frank, Joachim. Toward an Understanding
of the Structural Basis of Translation. Genome
Biology. 4237. 2003. Moore, Peter. The Ribosome
at Atomic Resolution. Biochemistry. 40 (11).
2001. Nierhaus, Wadzak, et al. Structure of the
elongating ribosome Arrangement of the two tRNAs
before and after translocation. Proc. Natl.
Acad. Sci. 95, 1998.
4 Translocation occurs. The used tRNA leaves
from the E site and the process repeats until a
stop codon is reached
Termination
When the ribosome reaches a stop codon, release
factors resembling tRNA bind to the A site and
the nascent peptide chain is released