Protein Synthesis 1
Major topics covered:
•The genetic code
•tRNA: aminoacylation and base-pairing
•Ribosome structure/function: prokaryotic versus eukaryotic
related text:
Biochemistry
Garret and Grisham, 4th ed.
Chapter 30
contact info:
David A. Schneider, Ph.D.
Department of Biochemistry and Molecular Genetics
dschneid@uab.edu
office #: 934-4781
The Central Dogma of Biology:
DNA
RNA
protein
The Central Dogma of Biology:
DNA
Other
macromolecules
RNA
protein
The Central Dogma of Biology:
DNA
RNA
protein
A major molecular problem:
How do you take a 4-base DNA/RNA
code and interpret the instructions to
build proteins from a 20 amino acid
pool?
A major molecular problem:
How do you take a 4-base DNA/RNA
code and interpret the instructions to
build proteins from a 20 amino acid
pool?
rephrase:
How do you translate the 4-base
DNA/RNA language into appropriate
proteins?
Francis Crick proposed/predicted the
Adaptor Hypothesis
Crick’s Predictions:
– “…the RNA of the microsomal
particles, regularly arranged, is the
template”
– “…whatever went into the template
in a specific way did so by forming
hydrogen bonds”
– “…the amino acid is carried to the
template by an adaptor...”
– “such adaptors…might contain
nucleotides”
– “…a separate enzyme would be
required to join each adaptor to its
own amino acid…”
– “…the specificity required to
distinguish between … isoleucine
and valine would be provided by
these enzymes”
Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.
Currently Known As:
mRNA
Codon-Anticodon
Interactions
Aminoacyl-tRNA
tRNA
Aminoacyl-tRNA
Synthetase
Editing by Aminoacyl-tRNA
synthetases
A visual model for the adapter hypothesis
Thus:
•Genes are codes (recipes, in a way)
•RNA polymerases copy the code into useful templates
•Translation (a collaboration of tRNA and ribosomes)
must crack the code correctly
The genetic code uses 3-base codons to generate
64 possible codon:anticodon interactions
(from the 4-base DNA/RNA sequence)
Different amino acids are encoded by
one or more codons
tRNAs are the adapters that “crack” the triplet
code and mediate the codon:anticodon pairing
Translation (on the surface) is very simple:
•Charged tRNAs bind to the appropriate codons
•Put a bunch in a row, according to the recipe in the mRNA
•Bind all the amino acids together and, Wa-La!
Translation (on the surface) is very simple:
•Charged tRNAs bind to the appropriate codons
•Put a bunch in a row, according to the recipe in the mRNA
•Bind all the amino acids together and, Wa-La!
There are at least 3 major issues:
1. Proper amino acid must be attached to every tRNA
2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur
3. Triplet code must be interpreted in the proper frame
Problem #1
Charging of the tRNA (ie. aminoacylation)
The amino acid is covalently attached to the 3’
“acceptor stem” of the tRNA by proteins called tRNA
synthetases
tRNA cloverleaf diagram
tRNAGln bound to glutaminyl-tRNAGln synthetase
Aminoacylation occurs by one of two pathways
(class I or class II)
The interaction between the tRNA, the
appropriate amino acid and the tRNA
synthetase is exceptionally important for
translational fidelity
The structure of tRNAGln bound to its cognate
tRNA synthetase demonstrates one mechanism
for specificity
Diagram of tRNA “identity elements”
Identity elements in tRNAs
Size of yellow ball
is proportional to the
fraction of 20 tRNA
acceptor types for which
the nucleoside is an
observed determinant
tRNA synthetases can edit incorrect
aminoacylation events as well
There are at least 3 major issues:
1. Proper amino acid must be attached to every tRNA
2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur
3. Triplet code must be interpreted in the proper frame
Problem #1 is solved!
How do the appropriate tRNAs bind
to the correct triplet codon?
Codon : Anticodon binding specificity
Base pairing rules for the THIRD position of the codon
Illustration of non-specific
interactions with inosine
A “wobble” example
Codon:
5’-CAC-3’
Anticodon: 3’-GUG-5’
Codon:
5’-CAU-3’
Anticodon: 3’-GUG-5’
There are at least 3 major issues:
1. Proper amino acid must be attached to every tRNA
2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur
3. Triplet code must be interpreted in the proper frame
Problem #1 is solved!
and
Problem #2 is solved
How does translation choose the
correct reading frame of the
triplet code?
The “reading frame” problem, illustrated:
In this case, the solution is easy:
•Specific initiation of translation at a 5’ methionyltRNA codon (AUG)
•Strict, 3-nucleotide transitions during translation
elongation
There are at least 3 major issues:
1. Proper amino acid must be attached to every tRNA
2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur
3. Triplet code must be interpreted in the proper frame
All three problems are solved…
There are at least 3 major issues:
1. Proper amino acid must be attached to every tRNA
2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur
3. Triplet code must be interpreted in the proper frame
All three problems are solved…
Now:
What molecular machine executes
the process of translation?
The ribosome and translation
Topics covered in this portion of the lecture
(the rest of today and Monday):
•Prokaryotic ribosome structure
•Prokaryotic translation
•Prokaryotic versus Eukaryotic:
Ribosome features
Translation mechanisms
•Two examples of medical impact of translation
The prokaryotic ribosome structure has been
solved at atomic resolution
The bacterial ribosome is:
•2 subunits (50S and 30S)
•3 ribosomal RNAs (rRNAs)
•52 proteins
•Total Mass = ~2.5 million Daltons
A low resolution “structure” to understand
organization of sites in the ribosome
Alberts 6-64d?
A low resolution “structure” to understand
organization of sites in the ribosome
Alberts 6-64d?
How did the field progress from this cartoon to understanding
molecular details of this massive cellular machine?
Early cryo-electron
microscopy
experiments revealed
the general shape of
the ribosome:
led to initial
nomenclature
Better techniques led to better
models:
Three dimensional model of the
70S ribosome
CP, central protuberence
SP, spur
Cate et al. (1999) Science 285:2097.
Better EM models permit visualization of the functional
center of the 70S ribosome
Aminoacyl
Peptidyl
Exit
A P
Liljas (1999) Science 285:2077.
E
E
P A
The crystal structure of the prokaryotic large subunit
Large (50S) Subunit
•Proteins-purple
•23S rRNA-orange & white
•5S rRNA (top)-burgundy & white
•A site tRNA- green
•P site tRNA- red
From Cech, Science 289: 878 (2000)
The crystal structure of the prokaryotic large subunit
Large (50S) Subunit
•Proteins-purple
•23S rRNA-orange & white
•5S rRNA (top)-burgundy & white
•A site tRNA- green
•P site tRNA- red
No protein
sidechain atoms lies
within 18 angstroms
of the peptidyl
transferase site, so
ribosome is
officially a
ribozyme.
From Cech, Science 289: 878 (2000)
The crystal structure of the prokaryotic small subunit
Head
The small (30S) subunit:
Nose
•RNA = gold ribbon
•Proteins = colored ribbons
Platform
Shoulder
Body
Foot
Schluenzen et al., Cell 102: 615 (2000)
Functional sites mapped onto spacefill model of
large and small subunits
Green = A site
Blue = P site
Yellow = E site
2009 Nobel Prize in Chemistry was awarded for
structural insights into ribosome function
From left to right:
Venkatraman Ramakrishnan
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Thomas A. Steitz
Yale University, New Haven, CT, USA
Ada E. Yonath
Weizmann Institute of Science, Rehovot, Israel
-picture from New York Times
That is the ribosome.
Nest question:
What is translation and how does
it work?
We will deal with that on
Monday!
THE END
-any questions?