Protein Metabolism

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Protein Metabolism

• The genetic code
• Protein synthesis (translation)
• Protein targeting degradation

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What are needed for protein synthesis? gt70
ribosomal proteins gt20 enzymes to activate amino
acid precursors gt12 proteins/enzymes for the
initiation, elongation termination of
polypeptides 100 enzymes for the final
processing of proteins gt40 tRNAs rRNAs The
most complex biosynthetic process!
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Three major advances for the understanding of
protein synthesis 1) Paul Zamecnik et al., 1950s
(p.1021) newly synthesized, radiolabeled proteins
are accumulated at small ribonucleoprotein
particle (i.e., ribosome) in liver.
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Three major advances for the understanding of
protein synthesis (contd) 2) Mahlon Hoagland
Zamecnik activated amino acids are attached to a
heat-stable soluble RNA (i.e., tRNA), forming
aminoacyl-tRNAs. Aminoacyl-tRNA synthetases are
involved.
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3) Francis Cricks adaptor hypothesis a small
nucleic acid (perhaps RNA) could serve the role
of an adaptor, one part binding a specific a.a.
and aother part recognizing the nt sequence
encoding that a.a. in the mRNA.
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The triplet, nonoverlapping code Insertion or
deletion mutations alter the sequence of
triplets. Adding/subtracting 3 nt leaves the
remaining triplet intact, providing evidence that
a codon has 3 nt.
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codon a triplet of nucleotides that codes for a
specific amino acid. reading frame established
by the first codon, then begins a new one every
3 nucleotide residues.
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The Genetic Code Was Cracked Using Artificial
mRNA Templates
Marshall Nirenberg, 1961 enzymatic methods to
synthesize poly(U) gt phenylalanine
poly(C) gt proline poly(A) gt lysine
Nirenberg Philip Leder, 1964 trinucleotides
induce specific binding of aminoacyl-tRNA to
ribosome. H. Gobind Khorana, 1960s chemical
methods to synthesize polynucleotides with
repeating sequences of 3 4 bases gt polypeptides
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Dictionary of amino acid code words as they
occur in mRNAs.
termination codon (red)
initiation codon (green)
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Each reading frame gives a different sequence of
codons, but only one is likely to encode a given
protein.
Open reading frame (ORF) a reading frame
without a termination codon among 50 or more
codons.
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Codon is degenerate an amino acid may be
specified by more than one codon.
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Alignment of the two RNAs is antiparallel
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Codon pairing relationships when the tRNA
anticodon contains inosinate
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Protein Metabolism

• The genetic code
• Protein synthesis (translation)
• Protein targeting degradation

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Wobble Allows Some tRNAs to Recognize More than
One Coden
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Reading frame and amino acid sequence
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Dictionary of amino acid code words as they
occur in mRNAs.
termination codon (red)
initiation codon (green)
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EF-Tu
EF-G
GDP (red)
C-terminus (green) mimics tRNA
tRNA (green)
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Overlapping Genes in Different Reading Frames Are
Found in Some Viral DNAs
fX174
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Overlapping Genes in Different Reading Frames Are
Found in Some Viral DNAs Genes within genes
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Protein Synthesis involves five stages
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The Ribosome Is a Complex Supramolecular Machine
Masayasu Nomura et al., 1960s (p.1037) both
ribosomal subunits can be broken down into
their RNA and protein components, then
reconstituted in vitro.
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Structure of the bacterial ribosome at
near-molecular resolution
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Ribosomal subunits are identified by their S
(Svedberg unit) values, sedimentation
coefficients that refer to their rate of
sedimentation in a centrifuge.
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The sequences of the rRNAs of many organisms have
been determined. Each has a specific
three-dimensional conformation featuring
extensive intrachain base pairing.
Models for the secondary structure of E. coli
16S and 5S rRNAs
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Transfer RNAs Have Characteristic Structure
Features
Robert H. Holley et al., 1965 (p.1038) yeast
tRNAAla
cloverleaf conformation ????
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General cloverleaf secondary structure of all
tRNAs
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Three-dimensional structure of yeast tRNAPhe
deduced from X-ray diffraction analysis
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Protein Synthesis

• Stage1 Aminoacyl-tRNA synthetases attach the
  correct amino acids to their tRNAs
• Stage 2 A specific amino acid initiates protein
  synthesis
• Stage 3 Peptide bonds are formed in the
  elongation stage

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Amino acid tRNA ATP
Mg2
Aminoacyl-tRNA synthetase
aminoacyl-tRNA AMP PPi
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Proofreading by aminoacyl-tRNA synthetase
e.g., Ile-tRNAIle synthetase favors activation of
Ile over Val by a factor of 200, i.e., it
distinguishes between Val and Ile.
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Interaction between an aminoacyl-tRNA synthetase
and a tRNA a second genetic code
Recognition sites by unique enzyme
(orange) several enzymes (green) all enzymes
(blue)
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Gln-tRNA synthetase
Asp-tRNA synthetase (dimeric)
tRNA (green) bound ATP (red)
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The tRNAAla elements recognized by the Ala-tRNA
synthetase are usually simple. Just a single GU
base pair (red)!
Synthetic simple form also works!
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Protein Synthesis

• Stage1 Aminoacyl-tRNA synthetases attach the
  correct amino acids to their tRNAs
• Stage 2 A specific amino acid initiates protein
  synthesis
• Stage 3 Peptide bonds are formed in the
  elongation stage

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Howard Dintzis, 1961 polypeptides grow by
addition of new amino acid to the carboxyl end
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synthetase
Met tRNAfMet ATP Met-tRNAfMet AMP
PPi N10-Formyltetrahydrofolate
met-tRNAfMet tetrahydrofolate fMet-tRNAfMet
transformylase
tRNA
The distinction between initiating AUG and
internal one is straightforward...
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Three steps of initiation
Aminoacyl site Peptidyl site Initiation Factor
(IF)
the initiation complex forms in the expense of
the hydolysis of GTP to form GDP and Pi.
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The initiating AUG is guided by the
Shine-Dalgarno sequence in the mRNA
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Protein complexes in the formation of a
eukaryotic intiation complex
eIF
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Protein Synthesis

• Stage1 Aminoacyl-tRNA synthetases attach the
  correct amino acids to their tRNAs
• Stage 2 A specific amino acid initiates protein
  synthesis
• Stage 3 Peptide bonds are formed in the
  elongation stage

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Peptide Bonds Are Formed in the Elongation Stage

• Elongation requires
• the initiation complex
• aminoacyl-tRNAs
• elongation factor (EF-Tu, -Ts,-G)
• GTP

Proofreading on the ribosome EF-Tu.GTP/EF-Tu.GDP
complexes (milliseconds) provide
opportunities for the codon-anticodon
interactions.
Elongation step 1 Binding of the second
aminoacyl-tRNA
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Elongation step 2 Formation of the first
peptide bond
Peptide transferase
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Elongation step 3 Translocation
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EF-Tu/tRNA EF-G/GDP
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How to know GTP is involved? GTP analog slows
hydrolysis, improving the fidelity (by increasing
the proofreading intervals) but reducing the
rate of protein synthesis.
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Termination of polypeptide synthesis requires a
special signal
Release (or termination) factor
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Energy cost of fidelity in protein synthesis
More than 4 high-energy bonds are required for
the formation of each peptide bond of a
polypeptide 2 ATP/GTP during aminoacyl-tRNA
formation 2 GTP during the first elongation step
translocation
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Rapid translation of a single mRNA by polysome in
both prok./euk. cells
Polysome a fiber between adjacent ribosomes in
the cluster of 10 to 100.
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A polysome from the silk gland of a silkworm
larva
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Coupling of transcription and translation in
bacteria
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Posttranslational modification
e.g., modification of individual amino acids
phosphorylation
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e.g., modification of individual amino acids
carboxylation
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e.g., modification of individual amino acids
methylation
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Posttranslational modification (contd)
e.g., addition of isoprenyl groups
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Protein Synthesis Is Inhibited by Many
Antibiotics and Toxins
e.g., disruption of peptide bond
formation by puromycin
Peptidyl puromycin
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Protein Metabolism

• The genetic code
• Protein synthesis (translation)
• Protein targeting degradation

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George Palade... David Sabatini Gunter Blobel,
1970
Signal sequences of some eukaryotic proteins
Signal recognition particle (SRP)
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Directing eukaryotic proteins with the
appropriate signals to the endoplasmic reticulum
(ER)
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Glycosylation Plays a Key Role in Protein
Targeting
Synthesis of the core oligosaccharide of
glycoproteins
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Tunicamycin mimics the structure
of UDP-N-acetylglucosamine (UDP-GlcNAc) and
blocks the first step of glycosylation.
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Phosphorylation of mannose on lysosome-targeted
enzymes, e.g., hydrolase
M-6-P
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Proteins Are Targeted to Mitochondria and
Chloroplasts by Similar Pathways
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Signal Sequences for Nuclear Transport Are Not
Cleaved
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Bacteria Also Use Signal Sequences for Protein
Targeting
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Model for protein export in bacteria
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Cells Import Proteins by Receptor-Mediated
Endocytosis
clathrin
coated pit
Joe Goldstein Mike Brown
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Protein Degradation Is Mediated by Specialized
Systems in All Cells Proteasome (Mr 1 x
106) The ATP-dependent, ubiquitin involved
proteolytic system in eukaryotes. Three-step
cascade pathway by which ubiquitin is attached
to a protein.
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Three-dimensional structure of the eukaryotic
proteasome
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The Nobel Prize in Chemistry 2004
"for the discovery of ubiquitin-mediated protein
degradation"
Aaron Ciechanove
Avram Hershko
Irwin Rose