Download The Ribosome, rRNA and mRNA (3.1)

The Ribosome, rRNA and mRNA (3.1)
Lecture 3
The Ribosome, rRNA and mRNA
Major players in protein synthesis:
mRNA, tRNA and the ribosome
mRNA
Messenger RNA, a copy of DNA blueprint of
the gene to be expressed.
tRNA
Aminoacyl transfer RNA, also called anticodon
or adaptor molecule. One or more tRNAs for
each amino acid.
Supply
Ribosome
A very large complex of several rRNAs
(ribosomal RNA) and many protein molecules.
Total molecular weight almost 3 million dalton.
Factory
Protein
Polypeptide chain with sequence dictated by the
mRNA sequence. Also called the gene product.
Product
The Ribosome
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Information
The Ribosome, rRNA and mRNA (3.1)
Ribosomes can be found either free in the cytosol
(cytoplasm) or attached to intracellular membranes.
Free ribosomes
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Found in the cytosol.
May occur as a single ribosome or in groups known as polyribosomes or polysomes.
Occur in greater number than bound ribosomes in cells that retain most of their manufactured protein.
Responsible for proteins that go into solution in the cytoplasm or form important cytoplasmic structural
or motile elements.
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The Ribosome, rRNA and mRNA (3.1)
Bound ribosomes
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Found bound to the exterior of the endoplasmic reticulum (ER) constituting the rough ER.
Occur in greater number than free ribosomes in cells that secrete their manufactured proteins (e.g.,
pancreatic cells, producers of digestive enzymes).
Responsible for proteins that insert into membranes or are packaged into vesicles for storage in the
cytoplasm or export to the cell exterior.
Electronmicrograph of ribosomes (black dots) attached to the rough endoplasmic reticulum.
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About 15,000 ribosomes in a single E. coli cell, comprising ~25% of the dry cell mass.
All ribosomes within one cell are identical.
All ribosomes are composed of two subunits (called small and large).
A substantial fraction of ribosomes are dissociated into free subunits in the cell.
Prokaryotic and eukaryotic ribosomes differ in composition.
Mitochondrial ribosomes resemble prokaryotic ribosomes.
Next: The Composition of Ribosomes
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The Ribosome, rRNA and mRNA (3.1)
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The Composition of Ribosomes (3.2)
The Composition of Ribosomes
Prokaryotic ribosomes
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Sedimentation coefficient: 70S
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small subunit: 30S
■ One rRNA molecule (16S)
■ 21 different proteins, designated S1-S21
large subunit: 50S
■ Two rRNA molecules (5S and 23S)
■ 31 different proteins, designated L1-L31 (L12 is present in four copies)
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The Composition of Ribosomes (3.2)
The sedimentation coefficient is measured in Svedberg units (S): the rate of
sedimentation of a component in a centrifuge is related both to the molecular weight and
the 3-D shape of the component.
The rRNA components of the prokaryotic ribosome
Type
Approximate number
of nucleotides
Subunit location
16S
1,542
30S
5S
120
50S
23S
2,904
50S
RNA function and turnover in the cell
Nucleotides
% of
Synthesis
% of
Total
RNA
Thousands
500-6000
40-50
3
3 (23S, 16S,
5S)
2904, 1542, 120
50
90
Stable
~50
73-93
3
7
Stable
Different
Kinds
mRNA Messenger
rRNA Ribosome
Type
tRNA
Function
Adapter
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Stability
T1/2 = 1-3
min
The Composition of Ribosomes (3.2)
Separation of ribosomal proteins by 2D gel electrophoresis
(a) small (30S) subunit; (b) large (50S) subunit
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The Composition of Ribosomes (3.2)
In 1968, Dr. M. Nomura, a professor here at UCI, was the first to show that
the 30S subunit could be reassembled from the individual components.
He found that the order of addition of components was critical.
Eukaryotic ribosomes
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Sedimentation coefficient: 80S
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small subunit: 40S
■ One rRNA molecule (18S)
■ 33 different proteins, designated S1-S33
large subunit: 60S
■ Three rRNA molecules (5S, 5.8S, and 28S)
■ 50 different proteins, designated L1-L50
The rRNA components of the eukaryotic ribosome
Type
Approximate number
of nucleotides
Subunit location
18S
1,900
40S
5S
120
60S
5.8S
156
60S
4,700
60S
28S
Next: Simplified Overview of Translation
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Simplified Overview of Translation (3.3)
Simplified Overview of Translation
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
Rate of synthesis
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Elongation is the rate limiting step in protein synthesis
In E. coli at 37 degrees C:
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Simplified Overview of Translation (3.3)
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ribosome passes through 15 codons per second
300 amino acid polypeptide made in 20 seconds
15,000 ribosomes per cell can make 750 molecules of a 300 amino acid protein per
second
Next: Structure of the Ribosome
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The Structure of the Ribosome (3.4)
The Structure of the Ribosome
(Prokaryotic ribosomes)
Proposed secondary structure of 16S rRNA
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The Structure of the Ribosome (3.4)
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Many regions are self-complementary and capable of forming double helical segments
Secondary structure is more highly conserved than primary sequence, i.e.
complementary mutations evolve to maintain base paring.
3-Dimensional structure of the 70S ribosome
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The Structure of the Ribosome (3.4)
The large subunit has a tunnel about 10 nm long and 2.5 nm in diameter. This tunnel is
thought to be the channel that newly assembled polypeptide chains pass through on
their way out of the ribosome.
The two subunits interact very tightly and form the active site
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The Structure of the Ribosome (3.4)
Different views: in yellow the 30S (small) subunit;
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in blue the 50S (large) subunit.
The Structure of the Ribosome (3.4)
Landmarks of the 30S subunit: h, head; p, platform; ch, channel
presumed to be the conduit for mRNA; sp, spur.
Landmarks of the 50S subunit: CP, central protuberance; St, L7/L12
stalk; L1, L1 protein; IC, interface canyon; T, tunnel presumed to be the
conduit for the nascent polypeptide chain; T1 and T2, lower tunnel
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The Structure of the Ribosome (3.4)
segments, leading to alternative exit sites E1 and E2, respectively.
Next: The X-ray Structure
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The X-ray Structure of the Ribosome (3.4b)
The X-ray Structure of the Ribosome
Two back-to-back landmark papers in the journal Science (August 2000) provided a whole new level of information
of what ribosomes look like in detail, and specifically, how the crucial peptide bond formation is catalyzed:
The Complete Atomic Structure of the Large Ribosomal Subunit at
2.4 Å Resolution
Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz
The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have
determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes
2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in
the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant
everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most
of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that
reach into the subunit's interior.
Science (2000) 289, 905-920.
The Structural Basis of Ribosome Activity in Peptide Bond Synthesis
Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz
Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs,
we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are
contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain
atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to
resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same
general base role as histidine-57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation constant) required for A2486 to
perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried
phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has
significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.
Science (2000) 289, 920-930.
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The X-ray Structure of the Ribosome (3.4b)
The first paper provides a wealth of structural information on how the components of the large (50S) subunit are
arranged:
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The X-ray Structure of the Ribosome (3.4b)
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the rRNA (in gray) is forming the core
the ribosomal proteins (yellow) are mostly on the surface
It also shows the exit tunnel and suggests that not only an extended polpypeptide would fit through it, but also one in
an alpha-helical conformation:
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The X-ray Structure of the Ribosome (3.4b)
The second paper provides a detailed mechanism for peptide bond formation based on the x-ray structure. It
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The X-ray Structure of the Ribosome (3.4b)
highlights the central role of the rRNA, and specifically that of base A2486 (RIBOZYME!):
Next: Supplemental Material
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The X-ray Structure of the Ribosome (3.4b)
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Supplemental Material (3.5)
Supplemental Material
This part won't be on the final!
If you are interested how findings like these are presented in an original
research article, you should take a look at the 1995 paper in the journal
Nature:
"A model of protein synthesis based on cryo-electron microscopy of the
E. coli ribosome"
by Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R.,
Lata, R.K. and Agrawal, R.K.
Nature 376, 441-444 (1995).
And for comparison, check out the more recent back-to-back X-ray papers
in the journal Science:
"The Complete Atomic Structure of the Large Ribosomal Subunit at
2.4 Å Resolution"
by Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz
Science 289, 905-920 (2000).
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Supplemental Material (3.5)
"The Structural Basis of Ribosome Activity in Peptide Bond Synthesis"
by Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz.
Science 289, 920-930 (2000).
These issues are available online or as hardcopies at the UCI Science
Library.
Next: Summary
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Summary (3.6)
Summary
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Ribosomes are large and complex molecular precision machines.
Ribosomes occur free in the cytosol or attached to the endoplasmic
reticulum.
They contain two subunits, called small and large.
Both subunits are comprised of large rRNAs and many proteins.
rRNAs form the core and are stabilized by extended base-stacking &
base-pairing.
Most ribosomal proteins are located on the surface.
Peptide-bond formation is catalyzed one base of the 23S rRNA
(ribozyme) in the large subunit.
Ribosomes have tunnels, channels and cavities which accommodate
the various players during translation: mRNA, aminoacyl-tRNA,
nascent protein.
Next Lecture: Initiation of Protein Synthesis
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