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Stable β-sheet aggregates can form from many proteins, forming intertwined

cross-beta strands that have the potential to kill cells or damage tissues.
Which of the following is NOT true regarding these aggregates?

A. They form almost exclusively in the cells of the nervous system.

B. Different types of such aggregates can form from the same protein.

C. Their formation is associated with conditions such as Parkinson’s disease


and Kuru.

D. They can form spontaneously, but also can be triggered to form by an


infection with the same aggregate.

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?

A) Lane 1 = 100 kd Lane 2 = 25 + 75 kd


B) Lane 1 = 25 + 100 kd Lane 2 = 25 kd
C) Lane 1 = 75 kd Lane 2 = 75 kd
D) Lane 1 = 100 kd Lane 2 = 25 kd
E) Lane 1 = 25 kd Lane 2 = 25 kd
Alternative splicing: alternatively select the reading frames.
Processing of 3’ End of pre-mRNA
• Termination signs are transcribed into RNA and recognized proteins as RNA Pol
transcribes throuth them
• CstF (cleavage stimulating factor F) and CPSF (cleavage and processing specificity
factor) proteins assoc w/ RNA Pol tail transferred to RNA as it emerges
What enzyme is depicted in the following schematic drawing?

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

A polysome (or a polyribosome) is a group of two or more ribosomes


translating an mRNA sequence simultaneously

The polysomes will appear as beads on a string (each 'bead' represents


a ribosome ; the ‘string’ is the mRNA strand)

In prokaryotes, the polysomes may form while the mRNA is still being
transcribed from the DNA template

Ribosomes located at the 3’-end of the polysome cluster will have


longer polypeptide chains that those at the 5’-end
Final decision of the proteins after their synthesis

• All proteins begin being


synthesized on ribosomes in
the cytosol, except for the few
that synthesized on the
ribosomes of the
mitochondria and plastids.

• Their final destination


depends on their amino acid
sequence, which can contain
sorting signals that direct their
delivery to locations outside
the cytosol.
• The ER, Golgi apparatus, and lysosomes are all
members of a network of membranes, but
they are not continuous with one another.
Therefore, the membrane lipids and proteins
that are synthesized in the ER must be
transported through the network to their final
destination in membrane-bound vesicles.
• the Golgi apparatus functions as a molecular
assembly line in which membrane proteins
undergo extensive post-translational
modification. Many Golgi reactions involve the
addition of sugar residues to membrane
proteins and secreted proteins.
Lysosomes break down macromolecules into their constituent parts,
which are then recycled. These membrane-bound organelles contain
a variety of enzymes called hydrolases that can digest proteins,
nucleic acids, lipids, and complex sugars. The lumen of a lysosome is
more acidic than the cytoplasm. This environment activates the
hydrolases and confines their destructive work to the lysosome. In
plants and fungi, lysosomes are called acidic vacuoles
Lysosomes are formed by the fusion of vesicles that have budded
off from the trans-Golgi. The sorting system recognizes address
sequences in the hydrolytic enzymes and directs them to growing
lysosomes. In addition, vesicles that bud off from the plasma
membrane via endocytosis are also sent to lysosomes, where their
contents — fluid and molecules from the extracellular environment
— are processed. The process of endocytosis is an example of
reverse vesicle trafficking, and it plays an important role in
nutrition and immunity as well as membrane recycling. Lysosomes
break down and thus disarm many kinds of foreign and potentially
pathogenic materials that get into the cell through such
extracellular sampling (Figure 3).