Biology 9700/22 — October/November 2010

Background Concept

Proteins that a cell secretes, inserts into its plasma membrane, or sends to lysosomes travel along a fixed pathway through the endomembrane system. The pathway begins when the gene encoding the protein is transcribed into mRNA inside the nucleus. The mRNA is then exported to the cytoplasm and translated on ribosomes bound to the rough endoplasmic reticulum (RER), so the growing polypeptide enters the RER lumen. From there, transport vesicles bud off and carry the protein to the Golgi apparatus, where further modification occurs. Modified proteins are sorted at the trans face of the Golgi and packaged into secretory vesicles. These vesicles travel to the plasma membrane and release their contents to the outside of the cell by exocytosis. Every step has a strict logical dependency: nothing downstream can occur until the step before has happened.

Understanding the Question

Part (a)(i) presents five events — exocytosis, protein modification, secretory vesicle formation, transcription, and translation — and asks you to write a number from 1 to 5 in the second column of Table 1.1 to show the order in which they occur (1 = first). This is a 2-mark question: 1 mark for getting the first and last events right, and 1 mark for the correct middle order.

Approach

Identify the unambiguous bookends first, then fill in the middle. Transcription must be first (DNA must be copied into mRNA before anything else can occur), and exocytosis must be last (it is the final release step at the cell surface). The middle three — translation, protein modification, and secretory vesicle formation — follow the journey of the protein through the endomembrane system. Work forward from the mRNA: once mRNA is made, it is translated into a polypeptide; the polypeptide is then modified; once modified, it is packaged into a vesicle for transport to the membrane.

Step-by-Step Reasoning

1. Transcription = 1: DNA is located only in the nucleus (in eukaryotes), and transcription (DNA → mRNA) must occur before translation can begin. The mRNA then leaves the nucleus through a nuclear pore.
2. Translation = 2: The mRNA is translated on ribosomes attached to the rough ER. The newly synthesised polypeptide is threaded into the RER lumen. This happens immediately after mRNA export.
3. Protein modification = 3: Inside the RER lumen and then within the Golgi cisternae, the polypeptide is chemically modified (signal peptide cleavage, disulfide bond formation, glycosylation, etc.). Modification must precede packaging because the protein is processed while moving through these compartments.
4. Secretory vesicle formation = 4: At the trans face of the Golgi, the mature protein is sorted and packaged into a secretory vesicle that buds off into the cytoplasm.
5. Exocytosis = 5: The secretory vesicle is transported to the plasma membrane, fuses with it, and releases its contents to the cell exterior.

The mark scheme awards 1 mark for transcription = 1 and exocytosis = 5, and a second mark for the correct middle order (translation = 2, protein modification = 3, secretory vesicle formation = 4).

Key Takeaways

• The secretory pathway is: DNA → mRNA → polypeptide → modification → packaging in secretory vesicles → release by exocytosis.
• Transcription is always the first step; exocytosis is always the last.
• Modification occurs before packaging because the protein is processed as it moves through the RER and Golgi.

Common Mistakes

• Putting translation first (forgetting that mRNA must be produced first).
• Swapping the order of protein modification and secretory vesicle formation (modification must occur before packaging, because the protein is processed within the Golgi before being sorted into vesicles).
• Putting exocytosis before secretory vesicle formation (the vesicle must form before it can fuse with the plasma membrane).
• Confusing transcription and translation (transcription makes mRNA from DNA; translation makes protein from mRNA).

Things to Be Careful About

• "Protein modification" here refers collectively to all modifications between translation and packaging, not a single chemical event.
• "Secretory vesicle formation" refers specifically to vesicles budding from the trans face of the Golgi, not the transport vesicles that move protein from RER to Golgi.
• This is an ordering question, so partial credit is limited; check the entire sequence before moving on.

Background Concept

Each event in the secretory pathway occurs at a specific, identifiable location. The nucleus houses the cell's DNA, so transcription must occur there. The rough endoplasmic reticulum (RER) is studded with ribosomes, which is why it is "rough"; these bound ribosomes translate mRNAs encoding secreted, membrane-bound, and lysosomal proteins, so the growing polypeptide enters the RER lumen. The RER is also a major site of initial protein modification (e.g. signal peptide cleavage, disulfide bond formation, N-linked glycosylation). The Golgi apparatus is a stack of flattened, membrane-bound cisternae that further modifies proteins (e.g. trimming and extending sugar chains, sulfation, phosphorylation) and sorts them into vesicles at its trans face. Secretory vesicles bud from the trans face of the Golgi and travel along cytoskeletal tracks to the plasma membrane, where they fuse with it and release their contents to the exterior by exocytosis. Lysosomes and smooth ER are not part of the secretory pathway: lysosomes digest material delivered to them, and smooth ER is involved in lipid synthesis and detoxification rather than protein processing.

Understanding the Question

Part (a)(ii) asks you to match each of the five events in Table 1.1 to a cell location from the list A to F:

• A — Golgi apparatus
• B — lysosome
• C — nucleus
• D — rough endoplasmic reticulum
• E — smooth endoplasmic reticulum
• F — plasma (cell surface) membrane

The instruction states that each letter may be used once, more than once, or not at all — a hint that some letters will be repeated and some will not be used at all. This is a 3-mark question.

Approach

For each event, identify the unique organelle where it occurs. Eliminate B and E first, because they are not involved in the secretory pathway for protein export. Then place each of the remaining five events at the appropriate location, remembering that protein modification can occur in more than one place.

Step-by-Step Reasoning

1. Transcription → C (nucleus): Transcription is the synthesis of mRNA from a DNA template. DNA is located only in the nucleus (in eukaryotes), so transcription occurs there.
2. Translation → D (rough ER): Translation of the mRNA occurs on ribosomes bound to the RER. The RER is rough specifically because of these attached ribosomes. Translation on free cytoplasmic ribosomes would produce a cytoplasmic protein, not a secreted one.
3. Protein modification → A or D (Golgi apparatus and/or RER): Modification occurs in both the RER (initial folding, signal peptide cleavage, disulfide bond formation, N-linked glycosylation) and the Golgi apparatus (further modification of sugar chains, sulfation, phosphorylation, sorting). The mark scheme accepts A, D, or A+D.
4. Secretory vesicle formation → A (Golgi apparatus): Secretory vesicles bud off from the trans face of the Golgi apparatus. The transport vesicles that move protein from RER to Golgi are not "secretory vesicles"; secretory vesicles specifically carry material destined for exocytosis.
5. Exocytosis → F (plasma membrane): Exocytosis is the fusion of a secretory vesicle with the plasma (cell surface) membrane, releasing the vesicle contents to the outside of the cell.

The mark scheme awards marks as follows: 1 mark for F, 1 mark for A or D (protein modification), and 1 mark for getting the other three letters (A for vesicle formation, C for transcription, D for translation) all correct.

Key Takeaways

• The nucleus is the site of transcription, not translation.
• The RER is the site of translation and of some early protein modification.
• The Golgi apparatus is the site of further modification and of secretory vesicle formation.
• Exocytosis occurs at the plasma membrane.
• Lysosomes and smooth ER are not part of the secretory pathway for protein export.

Common Mistakes

• Putting transcription in the cytoplasm (DNA is in the nucleus only).
• Putting translation on free ribosomes in the cytoplasm (this would produce a cytoplasmic protein, not a secreted one).
• Confusing the rough ER with the smooth ER (smooth ER is for lipid synthesis and detoxification, not protein synthesis).
• Putting secretory vesicle formation at the plasma membrane (vesicles form from the Golgi and then travel to the membrane).
• Putting protein modification only in the RER (it also occurs in the Golgi apparatus).

Things to Be Careful About

• Protein modification can be credited as A (Golgi), D (RER), or A+D (both).
• B (lysosome) and E (smooth ER) are not used in this secretory pathway.
• The mark scheme allows letters to be repeated (D appears for both translation and possibly modification), so do not be afraid to use the same letter for more than one event if the biology supports it.

Background Concept

DNA and RNA are polynucleotides. Each nucleotide carries one of four nitrogenous bases. DNA contains adenine (A), thymine (T), cytosine (C), and guanine (G). RNA contains adenine, cytosine, and guanine, but thymine is replaced by uracil (U). During transcription, RNA polymerase reads the DNA template strand in the 3′ → 5′ direction and synthesises a complementary mRNA strand in the 5′ → 3′ direction. The base-pairing rules for transcription are therefore:

• DNA A pairs with RNA U
• DNA T pairs with RNA A
• DNA C pairs with RNA G
• DNA G pairs with RNA C

The mRNA codon AUG is the universal start codon; it codes for the amino acid methionine and also signals the ribosome to begin translation at that point.

Understanding the Question

Part (c)(i) gives the DNA sequence TAC (this is the coding / non-template strand, which has the same sequence as the mRNA except with T in place of U). It asks for the mRNA sequence that is complementary to this DNA sequence. This is the mRNA codon for methionine. 1 mark.

Approach

Apply the base-pairing rules one base at a time, remembering that the mRNA will contain uracil (U) wherever the DNA contains adenine (A), because RNA has no thymine.

Step-by-Step Reasoning

• T (DNA) pairs with A (mRNA)
• A (DNA) pairs with U (mRNA) — note carefully: A in DNA pairs with U in mRNA, not with T, because mRNA contains uracil not thymine.
• C (DNA) pairs with G (mRNA)

So the mRNA codon is AUG.

Verification: AUG is the universal start codon in mRNA, which codes for methionine. This confirms the answer.

Key Takeaways

• The transcription base-pairing rules: DNA A → RNA U; DNA T → RNA A; DNA C → RNA G; DNA G → RNA C.
• A in DNA does NOT pair with T in mRNA (mRNA has no T); it pairs with U.
• AUG codes for methionine and is the universal start codon.

Common Mistakes

• Writing "ATG" instead of "AUG" (the most common error: forgetting that mRNA contains uracil, not thymine).
• Writing the complementary DNA sequence (treating it as if both strands were DNA).
• Reversing the order of bases (complementary base pairing preserves order; only the identities of the bases change).
• Pairing A with A, T with T, C with C, or G with G (these are not complementary pairs).

Things to Be Careful About

• The mRNA sequence must contain U, not T, because mRNA is RNA.
• Do not reverse the order of the bases — keep them in the same 5′ → 3′ direction as the DNA coding strand (read left to right).
• Commit AUG to memory as the methionine codon and the start codon.

Background Concept

A newly synthesised polypeptide chain is not yet a functional protein. After translation (and often during it), the polypeptide undergoes a series of post-translational modifications that convert it into its mature, active form. These modifications include:

1. Folding into secondary structure: the polypeptide backbone forms regular local patterns — $\alpha$-helices or $\beta$-pleated sheets — stabilised by hydrogen bonds between the backbone amide and carbonyl groups.
2. Folding into tertiary structure: the entire polypeptide folds into a specific three-dimensional shape, stabilised by interactions between the R groups of the amino acids (hydrogen bonds, ionic bonds, disulfide bridges between cysteine residues, and hydrophobic interactions that bury non-polar R groups in the protein interior).
3. Assembly of quaternary structure: two or more polypeptide chains (subunits) associate to form a functional protein. Examples include haemoglobin (four subunits) and antibodies (two heavy + two light chains).
4. Glycosylation: covalent addition of carbohydrate groups to the side chains of asparagine (N-linked) or serine/threonine (O-linked) residues, forming glycoproteins. This occurs in the RER and Golgi.
5. Addition of prosthetic groups: non-protein components essential for protein function, such as the haem group in haemoglobin, the iron-sulfur clusters in electron transport chain proteins, or metal ions (Fe, Cu, Mg, Zn) in many enzymes.
6. Proteolytic cleavage: removal of specific amino acids or cutting of the polypeptide into smaller pieces. Many hormones and enzymes are initially synthesised as inactive precursors (zymogens or pre-pro-proteins) that are activated by cleavage. Insulin, for example, is initially translated as a single chain (preproinsulin) and processed to a two-chain mature form held together by disulfide bonds.

Understanding the Question

Part (c)(ii) gives the example that methionine (the first amino acid) is removed to form a functioning protein. It asks you to suggest two OTHER ways in which the polypeptide chain is modified to produce the functioning protein. This is a 2-mark question: 1 mark for each correct, distinct modification.

Approach

Think about the four levels of protein structure (primary, secondary, tertiary, quaternary) and the additional chemical modifications that occur after translation. Choose two clearly different modifications from this list.

Step-by-Step Reasoning

The mark scheme accepts any two of the following (each valid for 1 mark):

1. Secondary structure: formation of $\alpha$-helices or $\beta$-pleated sheets.
2. Tertiary structure: folding into a 3D shape, stabilised by named bonds (hydrogen, ionic, disulfide).
3. Quaternary structure: assembly of multiple polypeptide subunits.
4. Glycosylation: addition of carbohydrates to form a glycoprotein. (The mark scheme rejects "hydrocarbon chain" because carbohydrates contain many OH groups, not hydrocarbons.)
5. Addition of a prosthetic group: e.g. haem, iron, copper, magnesium. These are non-protein components essential for the protein's function.
6. Removal of amino acids (more than one): e.g. cleavage of a signal peptide or of internal segments.
7. Polypeptide cleavage into two or more pieces: e.g. processing of preproinsulin to insulin.
8. AVP (any valid point): e.g. exposure of hydrophobic R groups to water during folding.

For 2 marks, give any two of the above as separate, clearly stated points.

Key Takeaways

• A newly synthesised polypeptide is not yet functional; it must undergo post-translational modifications.
• These modifications include folding (into 2°, 3°, 4° structures), glycosylation, addition of prosthetic groups, and proteolytic cleavage.
• Each modification is a specific, named chemical event — vague answers do not earn credit.

Common Mistakes

• Saying "the protein is folded" without specifying which level (primary, secondary, tertiary, quaternary) or what stabilises it.
• Listing only one modification when the question asks for two.
• Repeating the example given in the question (removal of methionine) — the mark scheme rejects the removal of just one amino acid as an "other" modification here.
• Saying "amino acids are joined by peptide bonds" (peptide bonds are formed during translation, not as a post-translational modification).
• Vague answers such as "the protein is changed" or "it becomes active" without giving a concrete mechanism.
• Writing "hydrocarbon chain" instead of "carbohydrate" for glycosylation.

Things to Be Careful About

• Each point must be a specific, named modification (e.g. "glycosylation", "addition of a prosthetic group", "formation of disulfide bonds").
• The mark scheme explicitly rejects peptide bonds, hydrocarbon chains, and removal of a single amino acid as valid answers here.
• For full marks, give two distinct modifications, not the same modification in two different wordings.