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cross-beta strands that have the potential to kill cells or damage tissues.
Which of the following is NOT true regarding these aggregates?
B. Different types of such aggregates can form from the same protein.
E. Some healthy cells form these aggregates to store their secretory proteins.
Question 1.2
You are studying a protein of 100 kilodaltons (kd). You found a way to cleave this
protein at a single site ¼ of the length from the N-terminus. You have raised a
monoclonal antibody that binds to the N-terminus of protein X.
You run on an SDS-PAGE a sample of intact protein X (on lane 1) and a sample of
protein X that has been cut at that single site (lane 2). If you do a Western Blot
analysis with the monoclonal antibody what will be the size of protein band(s)
observed in lanes 1 and 2?
3'
5'
5'
A. DNA polymerase
B. RNA polymerase
C. Ribosome
D. Reverse transcriptase
E. Topoisomerase
Processing the 3’ End of the Pre-mRNA
• Additional proteins assemble w/ CstF and CPSF to perform processing:
1. RNA is cleaved
2. Poly-A-Polymerase adds ~200 A’s to 3’ end of cleaved product
3. RNA Pol II continues to transcribe after pre- mRNA has been
cleaved; several 100 bases before falls off template and transcription terminates
4. RNA downstream of cleavage is degraded
Lecture 6 From RNA to Protein
1. Ribosome
2. tRNA
3. Translation
4. ER
5. Golgi apparatus
6. Lysosom
7. Polysome
5’ 3’
Strand to be
transcribed
An mRNA sequence is decoded in sets of
three nucleotides
Other
Triplet code
possibilities?
Ribosomes
Ribosomes are made of protein (for stability) and ribosomal RNA (for catalytic activity)
They consist of a large and small subunit:
The small subunit contains an mRNA binding site
The large subunit contains three tRNA binding sites – an aminoacyl (A) site, a peptidyl (P)
site and an exit (E) site
Ribosomes can be found either freely floating in the cytosol or bound to the rough ER (in
eukaryotes)
Ribosomes differ in size in prokaryotes and eukaryotes (prokaryotes = 70S (50S+40S) ;
eukaryotes = 80S (60S+40S))
Transfer RNA (tRNA)
tRNA molecules fold into a cloverleaf structure with four key regions:
The acceptor stem (3’-CCA) carries an amino acid
The anticodon associates with the mRNA codon (via complementary base pairing)
The T arm associates with the ribosome (via the E, P and A binding sites)
The D arm associates with the tRNA activating enzyme (responsible for adding the amino acid
to the acceptor stem)
1. Each tRNA molecule binds with a specific amino acid in the
cytoplasm in a reaction catalyzed by a tRNA-activating enzyme
2. Each amino acid is recognized by a specific enzyme (the enzyme may
recognize multiple tRNA molecules due to degeneracy)
3. The binding of an amino acid to the tRNA acceptor stem occurs as a
result of a two-step process:
4. The enzyme binds ATP to the amino acid to form an amino acid–
AMP complex linked by a high energy bond (PP released)
5. The amino acid is then coupled to tRNA and the AMP is released –
the tRNA molecule is now “charged” and ready for use
6. The function of the ATP (phosphorylation) is to create a high energy
bond that is transferred to the tRNA molecule
7. This stored energy will provide the majority of the energy required
for peptide bond formation during translation
Initiation
The first stage of translation involves the assembly of the three components that carry out the
process (mRNA, tRNA, ribosome)
The small ribosomal subunit binds to the 5’-end of the mRNA and moves along it until it
reaches the start codon (AUG)
Next, the appropriate tRNA molecule bind to the codon via its anticodon (according to
complementary base pairing)
Finally, the large ribosomal subunit aligns itself to the tRNA molecule at the P site and forms a
complex with the small subunit
• Elongation
• A second tRNA molecule pairs with the next
codon in the ribosomal A site
• The amino acid in the P site is covalently
attached via a peptide bond (condensation
reaction) to the amino acid in the A site
• The tRNA in the P site is now deacylated (no
amino acid), while the tRNA in the A site
carries the peptide chain
Translocation
The ribosome moves along the mRNA strand by one codon position (in a 5’ → 3’
direction)
The deacylated tRNA moves into the E site and is released, while the tRNA carrying the
peptide chain moves to the P site
Another tRNA molecules attaches to the next codon in the now unoccupied A site and
the process is repeated
Polysome
• Polysomes...
• A. are large cytoplasmic assemblies made of several
ribosomes each translating their exclusive mRNA.
• B. are only found in the eukaryotic cytoplasm.
• C. can take advantage of the circularization of
eukaryotic mRNA (by interactions between the 5' and 3'
ends of the mRNA) to further speed up the rate of
protein synthesis.
• D. are mostly translationally inactive and are normally
used by the cell to store the ribosomes and their
associated mRNAs for future use.
• E. All of the above.
Polysome
In prokaryotes, the polysomes may form while the mRNA is still being
transcribed from the DNA template