BCH 323 Tutorial 4

8th October 2019

A cartoon of a “typical” eukaryotic cell is shown below.

Endoplasmic
reticulum
5
2 1° RNA 3 4 Polypeptide
6 Functional
1 DNA mRNA
protein
nucleus 7
Amino acids

cytosol

QUESTION 1
Identify each of the processes labelled 1 to 7 and the major enzymes/molecules involved in these
processes. (20)

Step Process Enzymes/Molecules

1 DNA Replication
2 Transcription
3 mRNA Maturation and Translocation
4 Translation
5 Protein Sorting to ER
6 Post-translation Modifications
7 Protein Degradation
Orc1 to 6; RAP; RLFs; kinases – Cdc6 and
Cdt1; MCM/DNA helicase; Ddk; Mcm 10;
Cdc45; RFC; PCNA; DNA Dependent
DNA polymerases α, δ & ε; DNA ligase;
PCNA; FEN-1; RNase H1 and Telomerase
DNA Dependent RNA polymerase;
General Transcription Initiation Factors;
Enhancers/Silencers, Response elements;
CREB
Spliceome
Ribosome; mRNA; tRNA; Aminoacyl-
tRNA Synthetase; Peptidyl transferase
SRPs; Translocons; ATP/GTP; Hsp70s;
Signal peptidase
Kinases; Proteases;
Cathepsins; Peptide recognition protein;
Ubiquitin; Ubiquitin activating enzyme E1;
Ubiquiting conjugating enzyme E2;
Ubiquitin protein ligase E3; Proteasome
 QUESTION 2
Consider the mRNA sequence: 5’-A AUG CAG CUU CCU AGG UAG CA-3’.
(a) Write down the sequence of the double stranded DNA that this sequence was transcribed
from, indicating the polarity of each strand, which strand served as the coding strand
(template) and the coding region. (4)

5’-A ATG CAG CTT CCT AGG TAG CA-3’ => Coding strand

3’-T TAC GTC GAA GGA TCC ATC GT-5’ => Template strand

(b) Write down the amino acid sequence encoded by the mRNA using a coding table. (2)

AUG CAG CUU CCU AGG

Met – Gln – Leu – Pro - Arg

(c) How would the amino acid sequence change if the G from the second codon, GTC, in the
DNA template strand, is mutated to a A? (4)

5’-A ATG TAG CTT CCT AGG TAG CA-3’ => Coding strand

3’-T TAC ATC GAA GGA TCC ATC GT-5’ => Template strand

5’-A AUG UAG CUU CCU AGG UAG CA-3’

Met (Second codon in now a Stop Codon)

QUESTION 3
Describe in detail the two processes that may be involved in step 5, indicating the role/s of
relevant protein/s involved. (15)
Cotranslational Pathway:
Step 1: The ER signal sequence emerges from the ribosome,
Step 2: The ER signal it is bound by a signal-recognition particle (SRP).
Step 3: The SRP delivers the ribosome/nascent polypeptide complex to the SRP receptor in the ER
membrane. This interaction is strengthened by binding of GTP to both the SRP and its receptor.
Step 4: Transfer of the ribosome/nascent polypeptide to the translocon (Sec61 Complex) leads to
opening of this translocation channel and insertion of the signal sequence and adjacent segment of the
growing polypeptide into the central pore. Both the SRP and SRP receptor, once dissociated from the
translocon, hydrolyze their bound GTP and then are ready to initiate the insertion of another
polypeptide chain.
Step 5: As the polypeptide chain elongates, it passes through the translocon channel into the ER lumen,
where the signal sequence is cleaved by signal peptidase and is rapidly degraded.
Step 6: The peptide chain continues to elongate as the mRNA is translated toward the 3’ end. Because
the ribosome is attached to the translocon, the growing chain is extruded through the translocon into
the ER lumen.
Steps 7 & 8: Once translation is complete, the ribosome is released, the remainder of the protein is
drawn into the ER lumen, the translocon closes, and the protein assumes its native folded conformation.
Post-translational translocation ER (a).

Nascent polypeptides with signal sequences not recognized efficiently by SRP are bound by
Hsp70 Ssb (green, “B”) that is associated with ribosomes at the tunnel exit and/or soluble Hsp70
Ssa (gray, “A”).

• The Ssa C-terminal EEVD tetrapeptide is in red.

• Ssa and Ssb target nascent chains to SEC61 by binding to Sec72, a component of the
Sec62/63 complex (Sec62/63, dark gray).

• Ssa interacts via its C-terminal EEVD tetrapeptide, and Ssb via its nucleotide binding
domain. Not shown: J-proteins needed for Hsp70 binding to polypeptide substrate to
facilitate ATP hydrolysis and NEF for exchange of nucleotide and thus release of
substrate from Hsp70. 

Post-translational translocation Mitochondria (b):

• Tom20 (pink cylinder) and Tom70 (purple cylinder), components of the TOM complex
embedded in the outer membrane, are receptors for proteins destined for the inner
membrane and matrix. 

• Proteins that bind Tom20 typically have an N-terminal, cleavable targeting sequence
(cyan line segment). 

• Tom70 targeting sequences (orange line segment) are typically in the protein’s interior.
Tom70 also binds the EEVD of Ssa type Hsp70s, helping to target these polypeptides to
the TOM translocon

QUESTION 4
List the possible modifications that may occur in step 6 and explain why each of these processes
may be necessary for the proper functioning of a cell. (6)

 N-formylmethionine in prokaryotes is cleaved

 Specific bonds in precursors are cleaved, as for example, preproinsulin to proinsulin to
insulin
 Leader sequences are removed by specific proteases of the endoplasmic reticulum; the
Golgi apparatus then directs the finished protein to its final destination
 Proteins can be phosphorylated on Ser, Tyr and Thr
 factors such as heme groups may be attached
 disulfide bonds may be formed
 amino acids may be modified, as for example, conversion of proline to hydroxyproline
 other covalent modifications; e.g., addition of carbohydrates
QUESTION 5
Describe in detail the two processes involved in step 7 clearly indicating the function of each
process and the relevant proteins or molecules involved. (14)

[65]

Lysosomal Degradation Pathway

 Contain ~50 hydrolytic enzymes including proteases known as cathepsins. Acidic pH optima
and are inactive at cytosolic pH. Lysosomes recycle proteins taken up in autophagic
vacuoles or substances taken up by the cell through endocytosis.
 Under well-nourished conditions proteins are degraded non-selectively.
 Under fasting conditions, a 73 kD peptide recognition protein (prp73) binds to proteins
containing a KFERQ pentapeptide and delivers them to the lysosome for degradation.
 This selective degradation limits degradation to only those organs that undergo atrophy under
fasting conditions e.g. liver & kidneys and no degradation occurs in the brain or testes.

ATP-dependent protein degradation pathway.

 Ubiquitin, a 76 residue monomeric protein found ubiquitously and abundantly, is required.
 Proteins are selected for degradation by covalent linking with ubiquitin, which occurs in
three steps:
 Ubiquitin’s terminal carboxyl group is conjugated to ubiquitin-activating enzyme (E1), via a
thioester bond. Most organisms only have one E1.
 The ubiquitin is then transferred to a specific Cys sulphydryl group on one of numerous
ubiquitin-conjugating enzymes (E2).
 Ubiquitin-protein ligase (E3) transfers the activated ubiquitin from E2 to a Lys -amino
group of its target protein, forming an isopeptide bond. Cells contain many species of E3s,
each mediating the ubiquitination of a specific set of proteins, marking them for degradation.
E3s belong to either the HECT domain family or the RING finger family.
 Poly-Ubiquinated proteins are degraded by proteasome