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Biochemistry I
(CHE 418 / 5418)
Reading Assignment
Berg et. al (2007) Chapter 30
Central Dogma of Molecular Biology
Replication

DNA
Transcription
 RNA
Translation
 Protein
mRNA. tRNA, rRNA
• Replication - DNA directed DNA synthesis
• Transcription - DNA directed RNA synthesis
• Translation - RNA directed Protein synthesis
Translation
RNA directed protein synthesis
– Four letters of nucleic acid language translated into 20 amino
acids of protein language.
• mRNA, tRNA, ribosome (rRNA)
• Occurs on ribosome.
• Protein synthesized in N (amino) to C (carboxyl)
direction
• mRNA read from 5’ to 3’
N
5’
N
N
N
3’
Translation Divided into 3 Stages
Initiation
Elongation
Ribosome binds
mRNA identifies start
codon (AUG).
Termination
Protein synthesized
N to C
Polypeptide released
upon encountering
stop codons (UAA,
UAG, UGA)
* Met in Eukaryotes
* fMet N-formylmethionine in
prokaryotes
During Translation Ribosome binds:
• mRNA
– Contains codon – three
nucleotide sequence encoding
one amino acid.
• tRNAs (three sites)
– Contains Anticodon – three
nucleotide sequence
complementary to codon.
• Codon and Anticodon
interact by hydrogen bonding
between complementary
bases.
Ribosome contains three tRNA binding sites
• E – Exit
– Uncharged tRNA leaves
ribosome.
• P – Peptidyl
– tRNA in this site has protein
covalently attached.
• A – Aminoacyl
– Charged tRNA enters. Hbonding established between
codon (mRNA) and anticodon
of (tRNA).
Sites include regions of large and small subunits of ribosome.
Reaction to Add Amino Acids
• Amino acids are added to the C terminus by transferring
existing chain to next amino acid.
Polysome (Polyribosome)
• Polysome -multiple ribosomes
bond to the same mRNA.
– Each ribosome in a polysome is
synthesizing the same protein.
• In Prokayotes, transcription and
translation may be coupled, since
both are occurring in the same
compartment at the same time.
tRNA carry amino acids
• Amino acid (C
terminus) attached via
an ester to 2’or 3’
hydroxyl of A of CCA
on tRNA.
• “Charged tRNA” –
tRNA with correct
amino acid attached.
• Aminoacyl tRNA
synthetases – class of
enzymes that attach
amino acids to tRNA.
Aminoacyl tRNA synthetases
• Enzyme that attaches correct
amino acid to CCA of tRNA
with high fidelity. (>10-4).
• Binds Activation site and
anticodon.
• Two classes
– Class I
– Class II
Two Classes of
Aminoacyl
tRNA
synthetases
• Two classes differ in:
– Sequence alignment / comparison.
• 10 tRNA per class.
– Binding to tRNA on different
“face” or side
– Attaching amino acid to different
hydroxyl
• Class 1 to 2’ OH
• Class 2 to 3 ‘ OH
– Binding ATP in different
comformation
– Subunit comformation
• Class 1 are monomers
• Class II are dimers
Aminoacyl tRNA synthetases reaction
• Two step reaction
– 1. AA + ATP ↔ Aminoacyl-AMP + PPi
• Amino acid attached to AMP via mixed
anhydride which conserves the energy of
phosphodieaster bond in ATP.
• Acyl adenylate
– 2. Amino acid transferred to tRNA.
• Ester linkage to 2’ or 3’ hydroxyl of A of CCA
on 3’ end of tRNA.
Threonyl-tRNA synthetase
An example of Fidelity
• How does Threonyl-tRNA
synthetase select between
similarly shaped amino
acids?
• Thr has almost the same
shape as Ser and Val.
– 1. Active site selection
– 2. Editing site
Threonyl-tRNA synthetase
An example of Fidelity (Cont’)
• 1. Active site selection by
selective binding of
substrate.
– Zn 2+
• Coordinated to enzyme by 2
his and 1 cys residues
• Binds Thr by amino group and
OH of side chain
– Asp of enzyme
• Binds OH of thr substrate.
• This interaction prohibits
attachment of Val.
Threonyl-tRNA synthetase
An example of Fidelity (Cont’)
• 2. Editing site
– Removes Mischarged amino
acids (Ser).
• CCA with amino acid
attached swings into editing
site. Product does not
dissociate before editing.
• Increasing fidelity to
> 10-4
NOT ALL Aminoacyl tRNA
synthetases require editing.
Codon hydrogen bonds to Anticodon
• Codon of mRNA and
Anticodon of tRNA
are antiparellel.
Wobble
• Third base of
anticodon (5’ end)
allows binding with
multiple bases.
Codon Usage Table
• mRNA are “read” three nucleotides (codon) at a time
starting from a fixed point.
The Genetic code is unambiguous,
degenerate, non-overlapping and universal
• Unambiguous
– A given codon either designates a single amino acid or is
a stop codon.
• Degenerate
– more than one codon can specify the same amino acid,
so the genetic code is said to be degenerate.
• Non-overlapping
– the code is read sequentially, one codon after another
without spacer bases, from a fixed starting point.
• Universal
Initiation (Prokaryotic)
• Prokaryotic start codon =
AUG.
– Others exist (rare)
• GUG = Val
• UUG = Leu
• AUG encodes fMET
• Two important interactions
– 1. Binding of mRNA to 3’
end of 16S rRNA.
– 2. H bonding of initiator
codon with anticodon.
• tRNAf binds to P site of
ribosome.
Elongation (Prokaryotic)
• P site is occupied by tRNA holding fMet, or later the
growing polypeptide
• Elongation factors bring the correct charged tRNA to the
A site.
– EF-Tu – brings charged tRNA to A site of ribosome. Requires
bond GTP (hydrolyzed to GDP during reaction). GTP binds to
p-loop.
– EF-Ts – removes GDP from EF-Tu and replaces with GTP
• The 23 S rRNA of the ribosome catalyzes the attachment
of amino acids.
• Translocation is accelerated by EF-G.
Elongation (Prokaryotic)
Termination (Prokaryotic)
• Termination aided by
RF = release factors.
Castor Oil
• What is Castor Oil?
– Thick, yellowish or almost colorless oil
extracted from castor bean (seed).
• Uses of Castor Oil
–
–
–
–
–
–
–
Laxative
Lubricating oil
Quick-drying oil (when dehydrated)
used in paints and varnishes.
Competes with linseed and tung oil
Coating fabrics /protective coverings
Sebacic acid - basic ingredient in the
production of nylon 6, 10 and other
synthetic resins / fibers
Castor Bean
• Ricinus communis
– (family Euphorbiaceae)
• Source of Castor Oil
http://www.ansci.cornell.edu/plants/alphalist.html
http://waynesword.palomar.edu/plmar99.htm#flow
Synthesis of Nylon 6, 10
– Condensation Polymerization
– Sebacic acid + 1, 6-hexanediamine
– Sebacic acid
– 10-carbon dicarboxylic acid
– carboxylic group (COOH) at each end of the
molecule
– 1,6-hexanediamine
– 6-carbon molecule with an amino group (CNH2) at each end.
– Free carboxylic acid of Sebacic Acid
reacts with amino group of 1, 6hexamediamine
– water molecule is produced at each link.
What is Ricin?
• Protein (64 kda) found
in pulp from production
of castor oil.
– Heterodimer
• A chain
– 267 amino acid
residues.
– Inhibits protein
synthesis.
• B-chain
– 262 amino acid
residues.
– facilitates transport into
cell.
• Connected by disulfide
bridge.
Mode of Action of Ricin
• A chain
– N-glycosidase which removes
specific adenine (depurinates) 28 S
ribosomal RNA
• A-4323 in rat liver
– prevents the binding of an
elongation factor, leaving the
ribosome incapable of protein
synthesis.
– Extremely toxic
• 70 micrograms (equivalent to
weight of grain of salt) will kill a
160 pound man.
– Merck Index: An Encyclopedia
of Chemicals, Drugs, and
Biologicals (1997)
Many Antibiotics and Toxins
Inhibit Translation