Protein synthesis, D1.2

• What is gene expression?

  Gene expression is the process by which information carried by a gene has observable effects on an organism.

  The function of genes is to specify the sequence of amino acids in a specific polypeptide. The polypeptides produced either directly or indirectly determine the observable characteristics of an individual.

  Genes can be switched on and off- some genes may never be switched on during the life of the cell (e.g. insulin), and some genes may always be expressed.
• What is transcription?


  Transcription is the synthesis of a length of mRNA from the DNA base sequences that constitute a gene.

  It is the first stage in gene expression, and the key stage where genes can be switched on and off. It occurs in the cell's nucleus.
• What is the process of transcription?

  Helicase attaches to the DNA and unwinds and unzips the relevant part of the DNA helix. Only one DNA strand acts as the RNA template- the Antisense strand. The other is a sense strand.

  As RNA polymerase moves along the antisense strand, nucleotides from the nucleoplasm are assembled in a precise order (to bond with the nucleotides on the antisense strand) due to complimentary base pairing. The nucleotides are added in a 5-prime to 3-prime direction of the growing mRNA strand.

  The fully formed RNA peels off the DNA and leaves the nucleus via a nuclear pore.

  The DNA then rewinds.
• What is RNA polymerase, and what does it do?


  1) Binds to the DNA at the start of the gene that is being transcribed

  2) Unwinds the DNA double helix and separates it into sense and antisense strands.

  3) Moves along the antisense strand (template strand)

  4) Positions RNA nucleotides on the antisense strand through complimentary base pairing

  5) Links RNA nucleotides with phosphodiester bonds to form a continuous RNA strand.

  6) Detaches the assembled RNA strand from the antisense strand and reforms the DNA double helix.
• How does complimentary base pairing present itself in transcription?

  Because RNA has a base sequence complimentary to the antisense strand, its sequence is identical to the sense strand, except for one difference: uracil replaces thymine

  The copying of base sequences during transcription depends on complimentary base pairing. Cytosine will always pair with Guanine, while Thymine on the antisense strand pairs with Adenine on RNA, and Adenine on the antisense strand pairs with Uracil on DNA.
• Why are DNA templates stable?

  When RNA polymerase unwinds the DNA forming the sense and the antisense strands, the base sequences do not change.

  The bases are only briefly vulnerable to chemical changes that may cause mutation (when the two strands are parted)

  DNA may be transcribed many times during the lifetime of a cell. This is why their stability is essential. If mutations were common, then frequently used templates would accumulate mutations and the RNA copies would contain more and more errors.
• What is a transcriptome?

  The full range of RNA types made in a cell is known as its transcriptome. Different cells or tissue types have different transcriptomes. Over time, the transcriptome changes as the activity of the cell also changes.
• What is translation?


  Translation is the process of polypeptide synthesis from mRNA. An amino acid sequence is made from a base sequence, translating the genetic code.
• Where does translation occur?

  Translation occurs in the cytoplasm (at a ribosome). The RNA with the information necessary for translation travels from one place to another within the cell (from the nucleus to the cytoplasm in eukaryotic cells), so is known as messenger RNA (mRNA).
• What is the process of translation, in simple terms?

  1) A ribosome binds to the mRNA near the start codon

  2) tRNA with the complementary anticodon binds to the start codon held in place by the large subunit of the ribosome. It brings an amino acid with it.

  3) The ribosome 'slides' along the mRNA to 'read' the next codon

  4) Peptide bonds form between the amino acids brought by tRNA
• What is the role of mRNA in translation?

  Has a site for ribosome binding

  Carries the genetic code

  Has a sequence of codons specifying the amino acid sequence of the polypeptide

  Has a start and a stop codon to indicate the start and the end of translation

  Can be translated multiple times
• What is the role of tRNA in translation?

  Has an anticodon at one end (three bases)

  Has an attachment point at the other end- for the amino acid corresponding to the anticodon

  Every tRNA molecule has a distinctive shape that is recognised by a dedicated activating enzyme, which attaches the correct amino acid onto the tRNA
• What is the role of the ribosome in translation?

  Consist of large and small subunits

  Small subunit has a binding site for mRNA

  Large subunit has three binding sites for tRNA

  Large subunit has a catalytic site that makes peptide bonds between amino acids

  Work in groups- known as polyribosomes

  Each ribosome takes around 1 minute to travel along a molecule of mRNA
• What is a codon, and what is an anticodon?

  Codons are the three-base sequence of nitrogenous bases present in a row on mRNA. These nitrogenous bases are Adenine, Guanine, Cytosine, and Uracil (thymine is present in DNA). The complementary sequence of the codons triplets are known as anticodons.

  One mRNA contains numerous codons, but one tRNA has only one anticodon, that codes for an amino acid. So, the main aim of codon and anticodon is to produce proteins and express genetic information.
• What is the accuracy of translation dependent on?

  The accuracy of translation is dependent on complimentary base pairing.

  The three bases of an anticodon on a tRNA must be complimentary to the three bases of the codon on mRNA for the tRNA to be able to bind to the ribosome and deliver its amino acid in translation.

  A- U

  C- G
• What is rRNA?


  rRNA (ribosomal RNA)

  - Makes the bulk of the ribosome

  - Large folded molecule
• What is mRNA?


  mRNA (messenger RNA)

  - Carries genetic message from DNA in the nucleus to the ribosomes where proteins are synthesised

  - Long molecule, not folded

  - Variable in length and base sequence

  - Readily broken down and remade
• What is tRNA?


  tRNA (transfer RNA)

  - Collects amino acids and takes them to the ribosomes for protein assembly

  - Small

  - Forms a 'clover leaf' shape

  - One end carries and amino acid

  - One loop has a triplet of bases (anticodon) which determines what amino acid it carries
• What is the genetic code, and what are its properties?

  The sequence of bases is known as the genetic code.

  The genetic code is a triplet code. This means that three bases code for one amino acid. Each triplet is known as an anticodon on tRNA and a codon on mRNA. There are 64 different combinations of 3 bases.

  The genetic code is also universal. It is found in all organisms.

  It is degenerate- with more than one codon for each amino acid.

  It contains specific start and stop codons.

  Triplet

  Universal

  Degenerate

  Start/stop codons
• What is codon recognition, as the first step of translation?

  The ribosome's A site (Aminoacyl site) is exposed to the next mRNA codon.

  A tRNA with a complementary anticodon binds to the codon.

  This tRNA carries the correct amino acid specified by the codon.
• What is peptide bond formation, as the second step of translation?

  The ribosome catalyses a bond between the amino acid in the A site and the polypeptide chain in the P site.

  The growing polypeptide is transferred from the tRNA in the P site to the new amino acid on the tRNA in the A site.

  This reaction is catalysed by peptidyl transferase (an enzymatic activity of the ribosome).
• What is translocation, as the third step of translation?

  The ribosome shifts (moves) one codon along the mRNA in the 5' → 3' direction.

  The tRNA that was in the P site moves to the E site (Exit site) and is released.

  The tRNA that was in the A site moves to the P site, now carrying the growing polypeptide.

  The A site is now open for the next incoming tRNA.
• What is a mutation?

  A gene mutation is a change to the base sequence of a gene. A single base substitution changes an entire codon, which means that the codon may code for a completely different amino acid. This affects the structure of a polypeptide/protein
• What does the gene Hb code for?

  The gene Hb codes for the beta-globin polypeptide in haemoglobin.
• What is the mutation that causes sickle cell anaemia?

  Most humans have the allele HbA (GAG). If a mutation converts the sixth codon of the gene from GAG to GTG, a new allele is formed- HbS (GTG).

  When the HbS allele is transcribed, the mRNA produced has GUG as its sixth codon rather than GAG.

  When the mRNA is translated, this causes the sixth amino acid in the polypeptide to be valine rather than glutamic acid (which it should be).

  HbA hemoglobin: Glutamic acid (polar)

  HbS hemoglobin: Valine (non-polar)
• What is Sickle cell anaemia?

  Sickle cell disease is the most common inherited condition in the world. The mutation that causes it may be inherited y offspring if it occurs in a cell of the ovary or testis.

  This change affects the haemoglobin molecules when they are in tissues with low oxygen concentrations- they link together into chains and form bundles that are rigid enough to distort the RBCs into a sickle shape. When oxygen concentrations are higher, the RBCs revert to their normal shape.

  The sickle-shaped RBCs become trapped in blood capillaries, blocking them and reducing blood flow. This causes damage to tissues, and ass RBCs circulate from low to high oxygen concentrations, they become damaged. Therefore, the life of a RBC is as little as four days. The body cannot replace the damaged RBCs at a rapid enough rate so anaemia develops.
• What are the mutations that lead to sickle cell anaemia?

  T to A in DNA

  normal mRNA - GAG with anticodon CUC carrying GLU

  mutated mRNA - GUG with anticodon CAG carrying VAL
• Start codon

  AUG - also codes for MET
• What is tRNA activation?

  tRNA activating enzyme binds a specific amino acid to a specific tRNA molecule. This is due to the shape of the tRNA and the active site of the tRNA activating enzyme.

  There are twenty tRNA activating enzymes in total.
• peptidyl transferase

  helps the peptide bond between amino acids in translation form- between the p an the a site on the ribosome
• Why is protein synthesis faster in prokaryotes?

  • Prokaryotic transcription and translation occur simultaneously in the cytoplasm, whereas eukaryotes separate these processes (nucleus for transcription, cytoplasm for translation).
  • Prokaryotic mRNA is polycistronic (can encode multiple proteins from one mRNA strand), whereas eukaryotic mRNA is monocistronic (one mRNA = one protein).
  • Prokaryotic ribosomes (70S) are more efficient in translation than eukaryotic ribosomes (80S) due to simpler regulatory mechanisms.
• Elongation

  The polypeptide chain grows, one amino acid at a time.

  Consists of:

  Codon recognition

  Peptide bond formation

  Translocation
• Codon recognition step

  tRNA molecule carrying an amino acid enters the A site, and the anticodon of the tRNA pairs with the complimentary codon of the mRNA
• Peptide bond formation step

  Ribosome catalyses the formation of a peptide bond between the amino acid in the A site and the growing polypeptide chain in the P site.

  The growing peptide chain is temporarily transferred to the A site
• What facilitates the peptide bond formation step?

  rRNA in the large subunit, acting as a ribozyme
• Translocation


  The ribosome shifts one codon on the mRNA