Transcript Ribosome structural studies

Microscopy of Ribosome Structure and Function
Justin Levy, Roger Ndindjock, James Potter, Justin Quon, Sam Veihmeyer
Early Microscopy
Current Mechanism of Translation
In the late 50’s, 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.
1
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.
1 – Initiation factor binding
2
Electron Microscopy
3
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.
Neutron Diffraction
2 – Binding of tRNA with
associated factors to form
preinitiation complex
Small-Angle-Neutron-Scattering (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 ribosome’s atomic
structure.
3 – eIF4G/eIF4A binding to
preinitiation complex
4 – Association of preinitiation
factor with mRNA
4
Eukaryotic 80S Ribosome
Initiation
60S Subunit
5 – Small ribosome subunit
scans for start codon
5
40S Subunit
28S rRNA
5.8S rRNA
18S rRNA
5S rRNA
49 Ribosomal Proteins
6 – Associated proteins falls off small
subunit, large subunit attaches and
translation begins
6
33 Ribosomal Proteins
Large Subunit Atomic Structure
(From http://en.wikipedia.org/wiki/Image:Ribosome_50s.png)
(From http://www.emunix.emich.edu/~rwinning/genetics/pics/transl1.jpg)
X-Ray Crystallography
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
Elongation
1
2
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 (-185°C) instead of liquid nitrogen
(-195°C) because of increased heat capacity; N2(l) will boil off
in the process of freezing, permitting unwanted ice crystals to
form.
3
4
Termination
1 – Charged tRNAs (aminoacyl-tRNAs)
bind to elongation factor eEF-1 in the
presence of GTP
2 – The complex enters the
empty A-site on a ribosome
carrying an initiator Met-tRNA
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 – Translocation occurs. The
used tRNA leaves from the E
site and the process repeats
until a stop codon is reached
When the ribosome reaches a stop codon, release
factors resembling tRNA bind to the A site and the
nascent peptide chain is released
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.
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.
4:237. 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.