Download Structure and Functions of Ribosomes

Structure and Functions of
Ribosomes
Project Assignment
S-114.500 Principles for Biosystems of the Cell
Mailiina Turanlahti
01/12/2004
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
1
Agenda
ƒ
ƒ
ƒ
ƒ
ƒ
Introduction
Structure of Ribosomes
Functions of Ribosomes
Research Methods for the study of Ribosomes
Programs for Visualisation and Modelling
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
2
Introduction: What are Ribosomes
ƒ Small cytoplasmic organelles found in all prokaryotes and
eukaryotes in large numbers
ƒ Typically 10,000 ribosomes in a bacterial cell and many more in eukaryotic cells
ƒ Size: ~20 nm diameter
ƒ Major actors in protein synthesis
ƒ Different “versions”
ƒ 80S* in Eukaryotes
ƒ 70S in Prokaryotes
ƒ 55S in eukaryotic chloroplasts and mitochondria
ƒ May exist in two forms in Eukaryotes
ƒ Bound to endoplasmic reticulum, forming rough ER
ƒ Free floating in the cytoplasm
* S = Svedberg units: a measure of the rate of sedimentation of a component in a centrifuge
that is related both to the molecular weight and the three-dimensional shape of the component
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
3
Agenda
ƒ Introduction
ƒ Structure of Ribosomes
ƒ Functions of Ribosomes
ƒ Research Methods for the study of Ribosomes
ƒ Programs for Visualisation and Modelling
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
4
General Features of the Structure of
Ribosomes
ƒ
ƒ
ƒ
Constitute of a smaller and a larger subunit
Building blocks: ribosomal RNA (rRNA) and many proteins
Most of the volume is occupied by RNA
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
5
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
6
Prokaryotic Ribosomes
ƒ
ƒ
ƒ
Small ribosomal subunits have a
head and a base with an armlike
platform extending from one side.
The large subunit has a prominent
central protuberance, stalk, and ridge
extending from one side.
The large subunit has a tunnel about
10nm long and 2.5 nm in diameter,
that extends from the region
containing the A (aminoacyl) and P
(peptidyl) sites to the part of the
large subunit from which the newly
assembled polypeptide chain exits
the ribosome
01/12/04
Eukaryotic Ribosomes
ƒ Contains some additional features
on the small subunit, including
ƒ a bill that extends from the head on the
side opposite to the cleft
ƒ a set of lobes at the end of the subunit
opposite to the head
ƒ Has three rRNAs (28S, 5.8S and
5S) on the large subunit, as
opposed to two on the
prokaryotic one
ƒ Proteins differ slightly from one
organism to another. For instance,
that mammals have the largest
ribosomes, due to different
proteins
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
7
Agenda
ƒ Introduction
ƒ Structure of Ribosomes
ƒ Functions of Ribosomes
ƒ Research Methods for the study of Ribosomes
ƒ Programs for Visualisation and Modelling
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
8
The Function of Ribosomes: Protein
Synthesis, or Translation
ƒ
ƒ
Principle: with the ribosome acting as a controlling site, the
ordered arrangement of tRNAs along the mRNA strand
carrying the genetic message induces the correct order for the
corresponding amino acids to form the polypeptide
The translation process consists of three sub processes:
1. Formation of the initiation complex
2. Elongation of the polypeptide chain (one repetition of the steps a, b and c for
every amino acid incorporated into the protein being synthesized)
a) binding of the aminoacyl-tRNA
b) peptide bond formation
c) translocation
3. Termination
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti
9
Translation (1): Initiation
1.
2.
3.
4.
5.
A ribosome separates into large
and small subunits
Met-tRNA combines with GTP in
a side reaction involving an
initiation factor
Met-tRNA is added to the small
ribosomal subunit
The small subunit is added to the
mRNA in a reaction driven by
ATP hydrolysis; attachment takes
place at the 5' cap of the mRNA;
once attached, the small subunits
moves or "scans" along the
mRNA until it reaches the AUG
initiator codon
The large ribosomal subunit is
added driven by the hydrolysis of
GTP brought to the complex with
the initiator tRNA; elongation
follows
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 10
Translation (2): Elongation
1.
Aminoacyl-tRNA binds to the A
site
2. Peptide bond formation
3. Peptidyl-tRNA formed at the A
site by step two is transferred
from the A site to the P site
Peptide bond formation:
ƒ catalyzed on the ribosome by
peptidyl transferase
a) Adjacent aminoacyl-tRNAs bound to
the mRNA at the ribosome
b) following peptide bond formation, an
uncharged tRNA is in the P site and a
dipeptidyl-tRNA in the A site.
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 11
Translation (3): Termination
1.
2.
3.
4.
A stop codon is encountered at the A site which causes the release
factor to bind to the A site along with GTP instead of aminoacyltRNA
The release factor binds to the stop codon and the bond holding the
polypeptide chain to the tRNA site at the P site is hydrolyzed,
catalyzed by the peptidyl tranferase site of the large subunit.
Since there is no amino acid located at the A site, the hydrolysis
allows the polypeptide chain to be freed from the ribosome; with
the release of the
polypeptide, the
release factor is
ejected from the
A site, and the
empty tRNA is
ejected from
the P site
Ribosomal
components
separate
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 12
Agenda
ƒ Introduction
ƒ Structure of Ribosomes
ƒ Functions of Ribosomes
ƒ Research Methods for the study of
Ribosomes
ƒ Programs for Visualisation and Modelling
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 13
Cryo-EM maps
ƒ Technique for getting 3D structures of biological molecules from an
electron microscope
ƒ Gathers many images of the same particle in various orientations, and then
uses e.g. computed tomography to reconstruct the 3D structure
ƒ Usually 15 Ångström resolution, but with enough images (hundreds of
thousands) resolutions of 7 or 8 Å are attainable
Cryo-EM
reconstruction of
E. coli ribosome at
11.5 Å resolution.
(a) Small subunit;
(b) large subunit;
(c) assembled ribosome
Gabashvili, I.S., Penczek, P et al., Solution structure of the E. coli ribosome at 11.5 Å resolution. Cell, Vol. 100, 2000
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 14
NMR Spectroscopy
ƒ Enables the research of biological macromolecules, e.g.
ribosomal proteins
ƒ The protein must first be cloned, expressed and purified
ƒ Samples of the protein are enriched with a radioactive isotope of carbon or
nitrogen in a predetermined way.
ƒ A spectrometer is used to record resonations of amines and single atoms that
follow the enriched parts of the molecule.
ƒ The calculation of the protein structure is an optimisation
problem, where the minimum energy is what is looked for.
ƒ The goal is to identify the full range of structures that are consistent with the
distance and angle constraints derived from the NMR data, while making sure
that the structure has reasonable molecular geometry, and attains the minimum
value of the energy function.
ƒ The method enables researching both the structure and the
dynamics of ribosomal proteins
ƒ There are significant limitations for the size of the molecule to be researched, as
the NMR data is highly complex
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 15
01/12/04
X-Ray Crystallography
ƒ Enables solving structures to atomic resolution
ƒ The ribosome, constituting of more than 100,000 atoms, is the
most complex biological structure that is solved at this
resolution
ƒ Many approximations and guesses have to be made, though
ƒ Requires first the crystallization of the protein
ƒ Crystals are built of well ordered elements, which diffract light or hard X-rays in
this case, in a characteristic way.
ƒ The characteristic diffraction pattern allows in principle to determine the precise
location of each individual atom within the crystal, e.g. within the ribosomal
particle.
ƒ The most useful methods of research combine X-ray
crystallography and Cryo-EM, by “moulding” the X-ray data
into the Cryo-EM maps
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 16
Agenda
ƒ
ƒ
ƒ
ƒ
Introduction
Structure of Ribosomes
Functions of Ribosomes
Research Methods for the study of Ribosomes
ƒ Programs for Visualisation and
Modelling
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 17
01/12/04
Programs for Visualisation and
Modelling
ƒ Aim: to find suitable programs to illustrate the 3D-structure of
ribosomes and their parts
ƒ Requirement: support for some file format in which
information about ribosome structure was stored, and made
available on the Internet
ƒ The programs were searched on Structural Biology Software
Database.
ƒ Several different structures were viewed with each of the
programs
ƒ Three programs were tested
ƒ Biodesigner (not very successfully)
ƒ MDL Chime
ƒ Cn3D
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 18
MDL Chime
ƒ Runs in the browser
(IE or Netscape 5.0
or higher) as a plug-in
ƒ Enables watching
molecule(s) provided
as a PDB file by the
author of the viewed
web page
Prokaryotic ribosome viewed with Chime. The RNA
backbone is shown as pink wire, and proteins as beige
spheres.
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 19
Cn3D
ƒ Helper application that allows
viewing 3-D structures from
NCBI's Entrez retrieval
service
ƒ A search in Entrez retrieval
service gave 282
representations of ribosomal
structures (most of a single
protein, though)
ƒ ”Simultaneously displays
structure, sequence, and
alignment ”
Structure of the ribosomal 40S subunit from yeast
viewed with Cn3D
01/12/04
S114.500-Principles for biocyctems of the cell
Mailiina Turanlahti 20