Protein synthesis

1. Overview

A gene is a sequence of nucleotides that codes for the amino acid sequence of a specific polypeptide. The coded information in DNA is converted into a polypeptide in two stages: transcription, in which the base sequence of the gene is copied into messenger RNA (mRNA), and translation, in which that mRNA is decoded at a ribosome to join amino acids in the correct order.

A key idea runs through both stages: the genetic code is a triplet code that is non-overlapping. The bases are read in consecutive groups of three, so three bases = one codon = one amino acid. Keeping this in mind makes both translation and the effects of mutations much easier to follow.

In eukaryotes the first RNA copy (the primary transcript) is processed by removing introns and joining exons before it leaves the nucleus. A change to the base sequence of a gene is a gene mutation, and depending on its type it may or may not alter the polypeptide produced.

Key Definitions

• Transcription: the process in which the base sequence of one strand of a gene is copied into a complementary molecule of messenger RNA (mRNA).
• Translation: the process in which the codon sequence of mRNA is decoded at a ribosome to assemble amino acids in the correct order to form a polypeptide.
• Template (transcribed) strand: the strand of the DNA double helix that is read by RNA polymerase to make mRNA.
• Non-transcribed strand: the strand of the DNA double helix that is not used as the template during transcription.
• Codon: a sequence of three adjacent bases in mRNA that codes for one amino acid or for a stop signal.
• Anticodon: a sequence of three bases on a transfer RNA (tRNA) molecule that is complementary to, and pairs with, a codon on mRNA.
• Intron: a non-coding base sequence within a eukaryotic primary transcript that is removed during RNA processing.
• Exon: a coding base sequence that is retained and joined to other exons to form the final mRNA.
• Gene mutation: a change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide.

Content

Transcription

Transcription takes place in the nucleus. The enzyme RNA polymerase binds to the start of a gene and unwinds the double helix, breaking the hydrogen bonds between the two strands over a short region. Only one of the two strands is read: this is the template (transcribed) strand, while its partner is the non-transcribed strand.

RNA polymerase moves along the template strand and lines up free RNA nucleotides opposite it by complementary base pairing. The base-pairing rules are the same as in DNA except that uracil (U) pairs with adenine in place of thymine; cytosine still pairs with guanine. The enzyme then catalyses the formation of phosphodiester bonds between adjacent RNA nucleotides, building a single strand of mRNA. The mRNA is therefore complementary to the template strand and identical in base sequence to the non-transcribed strand (with U in place of T). When the gene has been copied, the mRNA detaches and the DNA double helix re-forms.

RNA processing in eukaryotes

In eukaryotes the molecule made directly by transcription is the primary transcript, and it is not yet usable. It contains coding sequences (exons) interrupted by non-coding sequences (introns). During RNA processing, the introns are cut out and the exons are joined together (a process called splicing) to produce the mature mRNA. Only this processed mRNA leaves the nucleus through a nuclear pore and travels to a ribosome. Removing introns is essential, because if they were translated they would add incorrect amino acids and disrupt the polypeptide.

Translation

Translation occurs at a ribosome, in the cytoplasm or on the rough endoplasmic reticulum. A ribosome is made of ribosomal RNA (rRNA) and protein, and it has binding sites that hold two tRNAs against two adjacent codons at the same time. The ribosome binds to the mRNA and reads its bases three at a time, as codons. Each codon codes for one amino acid (or for a stop signal), so the codon order on mRNA determines the amino acid order in the polypeptide. Amino acids are delivered by transfer RNA (tRNA) molecules: each tRNA carries a specific amino acid at one end and has a three-base anticodon at the other end.

Elongation is a repeating cycle. The ribosome holds two codons at a time, so it works through the mRNA as follows:

1. A tRNA whose anticodon is complementary to the codon being read binds to the mRNA at the ribosome by complementary base pairing, bringing its amino acid into place.
2. A second tRNA binds at the next codon, so two tRNAs sit side by side, each carrying its amino acid.
3. The ribosome catalyses the formation of a peptide bond between the two amino acids.
4. The first tRNA then leaves (to be reloaded with another amino acid in the cytoplasm).
5. The ribosome moves one codon along the mRNA, and the next tRNA binds.

Steps 1-5 repeat, so the polypeptide grows one amino acid at a time. The cycle continues until the ribosome reaches a stop codon, at which point the completed polypeptide is released.

Summary of roles

• RNA polymerase: joins RNA nucleotides together during transcription using the template strand.
• mRNA: carries the coded information from DNA in the nucleus to the ribosome.
• codons: groups of three mRNA bases, each specifying one amino acid.
• tRNA: brings a specific amino acid to the ribosome and base-pairs with the mRNA.
• anticodons: three tRNA bases complementary to a codon, ensuring the correct amino acid is added.
• ribosomes: hold the mRNA and tRNAs in place and catalyse peptide bond formation.

Gene mutations

A gene mutation is a change in the base sequence of a DNA molecule. Because the base sequence determines the codon sequence in mRNA, and so the amino acid sequence, a mutation may result in an altered polypeptide. There are three main types:

• Substitution: one base is replaced by a different base. This changes only one codon, so at most one amino acid is altered. The effect is often small. Sometimes the new codon codes for the same amino acid (because the genetic code is degenerate), so the polypeptide is unchanged.
• Deletion: one or more nucleotides are removed.
• Insertion: one or more extra nucleotides are added.

A deletion or insertion of one or two nucleotides causes a frameshift: because the bases are read in non-overlapping threes, removing or adding a base shifts the reading frame so that every codon after the mutation is changed. This usually produces a completely different and non-functional polypeptide, so deletions and insertions tend to have a much greater effect than a single substitution.

The table below summarises how each type tends to affect the polypeptide:

Mutation type	What happens to the DNA	Codons affected	Likely effect on polypeptide
Substitution	One base swapped for another	One codon only	At most one amino acid changed; may be no change if the code is degenerate
Deletion	One or more bases removed	Every codon after the mutation (frameshift)	Usually a completely different, non-functional polypeptide
Insertion	One or more extra bases added	Every codon after the mutation (frameshift)	Usually a completely different, non-functional polypeptide

Worked example

Exam-style question: In a length of mRNA the codon GAU codes for the amino acid aspartate. In one cell a single base substitution in the gene changes this mRNA codon to GAG, which codes for glutamate; in another cell a deletion removes one base from the same region. Explain why the substitution alters only one amino acid but the deletion is likely to change the whole polypeptide. [3]

Model answer:

• A substitution replaces one base, so only one codon is altered; this changes at most one amino acid (here aspartate to glutamate) and the rest of the codons are read normally.
• A deletion removes a base, so all the bases after it are read in a different reading frame (a frameshift).
• Therefore every codon after the deletion is changed, giving a completely different amino acid sequence and usually a non-functional polypeptide.

Worked example

Exam-style question: Part of the template (transcribed) strand of a gene has the base sequence T A C G A C A A A. (a) Write the base sequence of the mRNA transcribed from this strand. (b) Write the three tRNA anticodons that would pair with this mRNA. (c) An adenine (A) nucleotide is then inserted at the very start of the gene, so the template now begins A T A C G A C A A A. State the new mRNA sequence and explain what effect this insertion has on the polypeptide. [5]

Model answer:

• (a) The mRNA is complementary to the template strand, with U in place of T: template T A C G A C A A A gives mRNA A U G C U G U U U.
• (b) Each tRNA anticodon is complementary to its mRNA codon, so it has the same base sequence as the template strand (with U for T): U A C, G A C, A A A.
• (c) The inserted A is transcribed, so the new mRNA reads U A U G C U G U U U. Reading in groups of three from the start, every codon is now different from the original.
• Note that the first codon itself is changed: the original start codon A U G becomes U A U. A frameshift alters the codon at the insertion point too, not only the codons after it.
• This is a frameshift: the reading frame has shifted by one base, so the whole amino acid sequence is changed from the insertion onward, usually giving a non-functional polypeptide.

Key Equations

This topic is qualitative; there are no equations to learn. The one quantitative rule to remember is the triplet code introduced in the Overview: bases are read in non-overlapping groups of three (one codon = three bases = one amino acid).

Common Mistakes to Avoid

• Defining a gene vaguely as "a piece of DNA". State precisely that a gene is a sequence of nucleotides that codes for the amino acid sequence of a specific polypeptide.
• Saying the mRNA is "the same as" or "a copy of" the template strand. The mRNA is complementary to the template strand; it has the same sequence as the non-transcribed strand, but with uracil in place of thymine.
• Forgetting that RNA uses uracil. When pairing bases during transcription, adenine on the template pairs with uracil, not thymine.
• Mixing up codon and anticodon. The codon is on mRNA; the anticodon is on tRNA, and the two are complementary.
• Confusing introns and exons. Introns are non-coding and are removed; exons are coding and are kept and joined together (a memory aid: ex-ons stay and are expressed).
• Claiming every mutation changes the polypeptide. A substitution may code for the same amino acid (the code is degenerate), so the polypeptide can be unchanged.
• Treating all mutation types as equally harmful. A single substitution affects at most one amino acid, whereas a deletion or insertion causes a frameshift that alters every codon downstream.
• Reasoning from the wrong mechanism when given information about a drug. If a question states that a substance binds to ribosomes or tRNA, explain its effect on translation (protein synthesis) rather than on an unrelated process such as cell wall formation.

Exam Tips

• Be clear about where each stage happens: transcription and RNA processing in the nucleus, translation at a ribosome.
• In sequencing questions, work out the mRNA from the template strand and remember to swap T for U.
• Use the word complementary when describing base pairing between template and mRNA, and between codon and anticodon.
• For mutation questions, always name the type (substitution, deletion or insertion) and then state its likely effect, distinguishing a single-amino-acid change from a frameshift.
• Watch the command word here: "outline" wants the type plus its likely consequence in brief, while "explain" wants the mechanism behind it (for example, that the reading frame is shifted so every subsequent codon is changed). Pitch the detail to the marks available.
• Mention that the code being degenerate is the reason some substitutions have no effect on the polypeptide.
• When asked for the roles of the key molecules, give each one a distinct function (for example, mRNA carries the message; tRNA brings the amino acid; the ribosome joins them) rather than describing them all as "making protein".