Download Lecture 7 - Columbus Labs

Quiz
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Advanced Topics in RNA and DNA
DNA Microarrays
Aptamers
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Quantifying mRNA levels to asses
protein expression
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The DNA Microarray Experiment
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Application of DNA Microarrays
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Some applications of microarrays
• Identification of tumor markers
• Distinguish between clinically distinct
subgroups of leukemia, lymphoma, breast
cancer, and melanoma
• Alterations in gene expression in response
to therapeutics to identify genes involved
in sensitivity and resistance
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Aptamers
• DNA, RNA, or protein that bind a specific
target
• Can be engineered or natural occurring
• Riboswitches are naturally occuring
aptamers that regulate gene expression
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How do RNA aptamers work?
Ligand induces
conformational change
allowing translation
Ligand induces
conformational change
inhibiting translation
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Structures of RNA aptamers
Flavin
Mononucleotide
FMN
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FMN aptamer binding site
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The Genetic Code
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All the codons have meaning: 61
specify amino acids, and the other 3
are "nonsense" or "stop" codons
The code is unambiguous - only one
amino acid is indicated by each of the
61 codons
The code is degenerate - except for
Trp and Met, each amino acid is
coded by two or more codons
Codons representing the same or
similar amino acids are similar in
sequence
2nd base pyrimidine: usually nonpolar
amino acid
2nd base purine: usually polar or
charged aa
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Biochemists Break the Code
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Assignment of "codons" to their respective amino
acids was achieved by in vitro biochemistry
Marshall Nirenberg and Heinrich Matthaei showed
that poly-U produced polyphenylalanine in a cellfree solution from E. coli
Poly-A gave polylysine
Poly-C gave polyproline
Poly-G gave polyglycine
But what of others?
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The Nirenberg Experiment – deciphering the genetic code
Ribosome-free cell
extract with
radiolabeled single
amino acid
Ribosomes alone
Ribosome-free cell
extract with radiolabeled
single amino acid
Ribosomes
Trinucleotide
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The Genetic Code is Nearly Universal
Codon
Standard code
Mitochondrial code
UGA
Stop
Trp
UGG
Trp
Trp
AUA
Ile
Met
AUG
Met
Met
AGA
Arg
Stop
AGG
Arg
Stop
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tRNA
• The tRNA molecules translate the mRNA
codon to a protein sequence
• There is a tRNA for each amino acid
• tRNA synthetase (one for each amino
acid) load the amino acid onto the tRNA
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How does each tRNA syntetase recognize which
Amino acid to load each tRNA with?
tRNA synthetase recognition elements Single
letter
amino
acid
code
U70
G3
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Structure of tRNA synthetase/tRNA complexes
Threonyl-tRNA synthetase/tRNAThr
Glutaminyl-tRNA synthetase/tRNAGln
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tRNA synthetase classes
Feature
Class I enzymes
Class II enzymes
Structure of the enzyme
active site
Parallel β-sheet
Antiparallel β-sheet
Interaction with the tRNA
Minor groove of the acceptor stem
Major groove of the acceptor stem
Orientation of the bound
tRNA
V loop faces away from the enzyme
V loop faces the enzyme
Amino acid attachment
To the 2′-OH of the terminal nucleotide of
the tRNA
To the 3′-OH of the terminal nucleotide
of the tRNA
Enzymes for *
Arg, Cys, Gln, Glu, Ile, Leu, Lys1, Met,
Trp, Tyr, Val
Ala, Asn, Asp, Gly, His, LysII, Phe, Pro,
Thr, Ser
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Aminoacyl-tRNA synthetase reaction
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Ribosomes
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Getting oriented with the
ribosome
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Protein synthesis
• Initiation
– Binding of mRNA by small ribosomal subunit at the
mRNA ribosomal binding site (a purine rich
sequence)
– Association of initiator tRNA to the first codon
– Association of the large ribosomal subunit
• Elongation
– Synthesis of all peptide binds by successive
association of the appropriate (dictated by the
codons) tRNA molecules
• Termination
– At the stop codon, releasing factor 1 and 2 along with
GTP bind, the peptide chain is released from the
tRNA at the P site, the tRNA is moved to the E site
and the ribosome dissociates
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Initiation
• Initiator tRNA (fMet-tRNA) –
recognizes start (AUG) codon and
is unique because the N-terminal
is formylated
• Initiation requires initiation factors
(IFs) that assemble the ribosomal
initiation complex – IF-2 is a
GTPase that delivers the fMettRNA to the ribosome P site
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Mechanism of Protein Synthesis
Peptidyl transferase
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Peptidyl transferase
Mechanism is not thought to be catalyzed
by acid-base chemistry (reaction pH
independent)
Catalyzed by several other mechanisms
Proximity
Proton shuttle
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Protein synthesis overview
A bit more detail
Elongation requires GTPases
EF-Tu loads the aminoacyl-tRNA into
position A
EF-G pushes the tRNA in
position A into position P and
the tRNA in position P to E
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Termination
RF-1 and RF-2
Binding of RF1/RF2/GTP
complex triggers
Cleavage of the polypeptide
chain from the tRNA
and then
the dissociation of the ribosome
complex
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tmRNA
Bacterial
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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 purine-rich 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 reassociation of ribosomal subunits in the
absence of an initiation complex.
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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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