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Review
Protein Synthesis
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tmRNA – in bacteria
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Eukaryotic and Prokaryotic Protein Synthesis Differences
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1. Ribosomes. Eukaryotic ribosomes are larger. (Slide 29, lecture 4)
2. Initiator tRNA. In eukaryotes, the initiating amino acid is methionine
rather than N-formylmethionine. However, as in prokaryotes, a special
tRNA participates in initiation.
3. Initiation. The initiating codon in eukaryotes is always AUG.
Eukaryotes, in contrast with prokaryotes, do not use a specific purinerich sequence (RBS) on the 5′ side to distinguish initiator AUGs from
internal ones. Instead, the AUG nearest the 5′ end of mRNA is usually
selected as the start site. A 40S ribosome attaches to the cap at the 5′
end of eukaryotic mRNA and searches for an AUG codon by moving
step-by-step in the 3′ direction. The 5′ cap provides an easily
recognizable starting point.
4. Elongation and termination. Eukaryotic elongation factors EF1α
and EF1βγ are the counterparts of prokaryotic EF-Tu and EF-Ts. The
GTP form of EF1α delivers aminoacyl-tRNA to the A site of the
ribosome, and EF1βγ catalyzes the exchange of GTP for bound GDP.
Eukaryotic EF2 mediates GTP-driven translocation in much the same
way as does prokaryotic EF-G. Termination in eukaryotes is carried out
by a single release factor, eRF1, compared with two in prokaryotes.
Finally, eIF3, like its prokaryotic counterpart IF3, prevents the
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reassociation of ribosomal subunits in the absence of an initiation
complex.
Eukaryotic Initiation
Binding of 5’ cap of mRNA
Ratchet search of AUG – dependent on ATP
Association of ribosome
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Antibiotics inhibit protein synthesis
Antibiotic
Action
Streptomycin and
other
aminoglycosides
Inhibit initiation and cause misreading of mRNA (prokaryotes)
Tetracycline
Binds to the 30S subunit and inhibits binding of aminoacyl-tRNAs
(prokaryotes)
Chloramphenicol
Inhibits the peptidyl transferase activity of the 50S ribosomal subunit
(prokaryotes)
Cycloheximide
Inhibits the peptidyl transferase activity of the 60S ribosomal subunit
(eukaryotes)
Erythromycin
Binds to the 50S subunit and inhibits translocation (prokaryotes)
Puromycin
Causes premature chain termination by acting as an analog of aminoacyltRNA (prokaryotes and eukaryotes)
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Proteins
• Building blocks (amino acids) and
properties
• Primary, Secondary, Tertiary, and
Quaternary Structure
• Stabilizing forces of a protein fold
• Functions
• Enzymes
• Mechanisms of Catalysis
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Amino Acids
Building Blocks of Proteins
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Amino Acids Can Join Via Peptide Bonds
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20 Common Amino Acids
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Non-polar amino acids
Polar, uncharged amino acids
Acidic amino acids
Basic amino acids
When looking at these structures I want
you to think about potential interactions
that could stabilize the three dimensional
structure of proteins
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Long haul to identifying the 20 aa
1819
1820
1846
1865
1866
1869
1873 (1932)
1873 (1932)
1875
1881
Leucine
Glycine
Tyrosine
Serine
Glutamic Acid
Aspartic Acid
Asparagine
Glutamine
Alanine
Phenylalanine
1889
1890
1895
1896
1901
1901
1901
1903
1922
1936
Lysine
Cysteine
Arginine
Histidine
Valine
Proline
Trytophan
Isoleucine
Methionine
Threonine
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Hydrophobic Amino Acids
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Hydrophobic Amino Acids
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Polar, Uncharged Amino Acids
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Polar, Uncharged Amino Acids
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Acidic Amino Acids
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Basic Amino Acids
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Uncommon Amino Acids
We may see some of these in later chapters
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Hydroxylysine, hydroxyproline - collagen
Carboxyglutamate - blood-clotting proteins
Pyroglutamate - bacteriorhodopsin
Phosphorylated amino acids - signaling
device
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Amino Acids are Weak Polyprotic Acids
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pKa Values of the Amino Acids
• Alpha carboxyl group - pKa = 2
• Alpha amino group - pKa = 9
• These numbers are approximate, and can
be perturbed by the local protein
environment.
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pKa Values of the Amino Acid Side Chains
• Arginine, Arg, R: pKa(guanidino group) = 12.5
• Lysine, Lys, K: pKa = 10.5
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Aspartic Acid, Asp, D: pKa = 3.9
Glutamic Acid, Glu, E: pKa = 4.3
Cysteine, Cys, C: pKa = 8.3
Histidine, His, H: pKa = 6.0
• Serine, Ser, S: pKa = 13
• Threonine, Thr, T: pKa = 13
• Tyrosine, Tyr, Y: pKa = 10.1
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Titration of Glycine
pI = (pK1 + pK2)/2
pH where the net
charge of the
molecule is zero
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Titration of Glutamic Acid
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Titration of Lysine
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Stereochemistry of Amino Acids
• All but glycine have chiral Cα
• L-amino acids predominate in nature
• R,S-nomenclature system is superior, since amino acids like
isoleucine and threonine (with two chiral centers) can be named
unambiguously
SH > OH> NH2 > COOH > CHO > CH2OH > CH3
All amino acids have the Cα configuration
of S except for cysteine because of the
thiol group
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Spectroscopic Properties
• All amino acids absorb in infrared region
• Only Phe, Tyr, and Trp absorb UV
• Absorbance at 280 nm can be used to
quantify the concentration for amino acids
The ε is proportional to the number
of F, W, and Y in the protein
Therefore you can calculate a
theoretical ε and use Beer’s law to
calculate the concentration of the
protein
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