protein synthesis 1 major topics covered: the genetic code
DESCRIPTION
Protein Synthesis 1 Major topics covered: The genetic code tRNA : aminoacylation and base-pairing Ribosome structure/function: prokaryotic versus eukaryotic. c ontact info: David A. Schneider, Ph.D. Department of Biochemistry and Molecular Genetics [email protected] office #: 934-4781. - PowerPoint PPT PresentationTRANSCRIPT
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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 [email protected] #: 934-4781
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The Central Dogma of Biology:
DNA
RNA
protein
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The Central Dogma of Biology:
DNA
RNA
protein
Othermacromolecules
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The Central Dogma of Biology:
DNA
RNA
protein
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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?
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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?
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Francis Crick proposed/predicted the Adaptor Hypothesis
– “…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”
Currently Known As:
mRNA
Codon-Anticodon Interactions
Aminoacyl-tRNA
tRNA
Aminoacyl-tRNA Synthetase
Editing by Aminoacyl-tRNA synthetases
Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.
Crick’s Predictions:
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A visual model for the adapter hypothesis
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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
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The genetic code uses 3-base codons to generate 64 possible codon:anticodon interactions
(from the 4-base DNA/RNA sequence)
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Different amino acids are encoded by one or more codons
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tRNAs are the adapters that “crack” the triplet code and mediate the codon:anticodon pairing
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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!
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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 tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame
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Problem #1
Charging of the tRNA (ie. aminoacylation)
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The amino acid is covalently attached to the 3’ “acceptor stem” of the tRNA by proteins called tRNA
synthetasestRNAGln bound to glutaminyl-tRNAGln synthetasetRNA cloverleaf diagram
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Aminoacylation occurs by one of two pathways (class I or class II)
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The interaction between the tRNA, the appropriate amino acid and the tRNA
synthetase is exceptionally important for translational fidelity
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The structure of tRNAGln bound to its cognate tRNA synthetase demonstrates one mechanism for
specificity
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Identity elements in tRNAs
Size of yellow ballis proportional to the fraction of 20 tRNA
acceptor types for whichthe nucleoside is an
observed determinant
Diagram of tRNA “identity elements”
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tRNA synthetases can edit incorrect aminoacylation events as well
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There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame
Problem #1 is solved!
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How do the appropriate tRNAs bind to the correct triplet codon?
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Codon : Anticodon binding specificity
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Base pairing rules for the THIRD position of the codon
Illustration of non-specific interactions with inosine
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Codon: 5’-CAC-3’Anticodon: 3’-GUG-5’
Codon: 5’-CAU-3’Anticodon: 3’-GUG-5’
A “wobble” example
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There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame
Problem #1 is solved!and
Problem #2 is solved
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How does translation choose the correct reading frame of the
triplet code?
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The “reading frame” problem, illustrated:
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In this case, the solution is easy:•Specific initiation of translation at a 5’ methionyl-
tRNA codon (AUG)•Strict, 3-nucleotide transitions during translation
elongation
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There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame
All three problems are solved…
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There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame
All three problems are solved…
Now:What molecular machine executes
the process of translation?
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Topics covered in this portion of the lecture(the rest of today and Monday):
•Prokaryotic ribosome structure•Prokaryotic translation•Prokaryotic versus Eukaryotic:
Ribosome featuresTranslation mechanisms•Two examples of medical impact of translation
The ribosome and translation
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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
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Alberts 6-64d?
A low resolution “structure” to understand organization of sites in the ribosome
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Alberts 6-64d?
A low resolution “structure” to understand organization of sites in the ribosome
How did the field progress from this cartoon to understanding molecular details of this massive cellular machine?
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Early cryo-electron microscopy
experiments revealed the general shape of
the ribosome:led to initial
nomenclature
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Better techniques led to better models:
Three dimensional model of the 70S ribosome
CP, central protuberenceSP, spur
Cate et al. (1999) Science 285:2097.
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Better EM models permit visualization of the functional center of the 70S ribosome
Liljas (1999) Science 285:2077.
AminoacylPeptidyl
Exit
A PE
APE
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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
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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)
No protein sidechain atoms lies within 18 angstroms
of the peptidyl transferase site, so
ribosome is officially a ribozyme.
The crystal structure of the prokaryotic large subunit
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Schluenzen et al., Cell 102: 615 (2000)
Head
Platform
Body
Foot
Shoulder
NoseThe small (30S) subunit:
•RNA = gold ribbon•Proteins = colored ribbons
The crystal structure of the prokaryotic small subunit
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Functional sites mapped onto spacefill model of large and small subunits
Green = A siteBlue = P siteYellow = E site
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2009 Nobel Prize in Chemistry was awarded for structural insights into ribosome function
-picture from New York Times
From left to right:Venkatraman RamakrishnanMRC Laboratory of Molecular Biology, Cambridge, United KingdomThomas A. SteitzYale University, New Haven, CT, USAAda E. YonathWeizmann Institute of Science, Rehovot, Israel
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That is the ribosome.
Nest question:What is translation and how does
it work?
We will deal with that on Monday!
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THE END
-any questions?