Gene Expression: From Transcription to Translation

Gene Expression: From Transcription to Translation

Overview

• Translation is the RNA-directed synthesis of a polypeptide.
• In bacteria, translation occurs in the cytoplasm.
• In eukaryotes, translation occurs in the cytoplasm after transcription and RNA processing in the nucleus. This processing includes:
  • Addition of a 5' cap.
  • Addition of a 3' poly(A) tail.
  • Removal of introns.

Translation Process

• Translation involves converting the nucleotide sequence of mRNA into the amino acid sequence of a protein.
• mRNA carries the genetic code in the form of codons, which are sequences of three nucleotides.
• These codons are "read" and translated into amino acids.
• Example mRNA sequence: 5'-AUG UUC GAA GAC AAC A-3'
• Corresponding amino acid sequence: NH3+-Met-Ser-Lys-Thr-Thr-COO-

Molecular Components of Translation

• mRNA (messenger RNA) carries the genetic code.
• tRNA (transfer RNA) molecules bring the correct amino acids to the ribosome.
• Ribosomes, composed of ribosomal RNA (rRNA) and proteins, are the site of protein synthesis.

The Genetic Code

• In 1966, Marshall Nirenberg and Heinrich Matthaei cracked the universal genetic code.
• The genetic code is a triplet code, where each codon (three nucleotides) specifies an amino acid.
• The code is degenerate, meaning that multiple codons can code for the same amino acid.
• Examples of codons and corresponding amino acids:
  • UUU and UUC: Phenylalanine
  • UCU, UCC, UCA, UCG: Serine
  • UAU and UAC: Tyrosine
  • UGU and UGC: Cysteine
  • UAA, UAG, UGA: Stop codons
  • AUG: Methionine (start codon)
• The table illustrates the relationships between mRNA codons and amino acids.

Adapter Molecules: tRNA

• tRNA molecules serve as adapter molecules, holding amino acids and interacting with mRNA codons.
• Each tRNA has an amino acid attachment site and an anticodon that recognizes the complementary codon on the mRNA.
• For example, a tRNA carrying phenylalanine has an anticodon AAG that binds to the mRNA codon UUC.

Transfer RNA (tRNA) Structure

• tRNA has an amino acid attachment site where the amino acid is attached.
• It also has an anticodon that recognizes the complementary codon on the mRNA.
• Example: Phenylalanine tRNA has the anticodon AAG, which pairs with the mRNA codon UUC.

Charging tRNA

• tRNAs are "charged" with the appropriate amino acid by aminoacyl-tRNA synthetases.
• Aminoacyl-tRNA synthetases use ATP to covalently link the amino acid to the 3' end of the tRNA.
• This process results in an aminoacyl tRNA (charged tRNA).
• Equation: Amino acid + tRNA + ATP --> Aminoacyl-tRNA + AMP + PPi

Ribosomes

• Ribosomes are particles made of rRNA and associated proteins.
• They are the site of protein synthesis and catalyze the formation of peptide bonds.
• Ribosomes bind mRNA and tRNA to synthesize polypeptides and proteins.

Ribosomal RNA (rRNA)

• rRNA plays a crucial role in ribosome structure and function.
• Eukaryotic (mammalian) ribosomes consist of:
  • A large 60S subunit, containing 5S, 5.8S, and 28S rRNA and 49 ribosomal proteins.
  • A small 40S subunit, containing 18S rRNA and 33 ribosomal proteins.

Ribosome Binding Sites

• Ribosomes have three sites for tRNAs:
  • A (aminoacyl) site: binds the incoming aminoacyl tRNA.
  • P (peptidyl) site: holds the tRNA with the growing polypeptide chain.
  • E (exit) site: where the tRNA that has discharged its amino acid exits the ribosome.

Translation Stages

• Protein synthesis (translation) is a complex cellular activity.
• The process is similar in prokaryotes and eukaryotes.
• Translation is divided into three stages:
  • Initiation
  • Elongation
  • Termination
• Each stage involves factors that aid in the process:
  • IFs: Initiation factors
  • EFs: Elongation factors
  • Release factors

Initiation

• The first step involves bringing the small ribosomal subunit to the initiation codon.
• Initiation factors (IFs) help attach the small ribosomal subunit to the initiation codon, using energy from GTP.
• In prokaryotes, the Shine-Dalgarno sequence in the mRNA (5-10 bases upstream of the initiation codon) helps align the ribosome.
• The initiator tRNA (fMet-tRNA in prokaryotes, Met-tRNA in eukaryotes) binds to IF2 and the start codon (AUG).
• The start codon sets the reading frame.
• The large ribosomal subunit then binds, completing initiation.
• Eukaryotic translation initiation involves:
  • Formation of an initiation complex with CBP (cap-binding protein), initiation factors, the small ribosomal subunit, and initiator tRNA.
  • Binding of the initiation complex to the 5' cap of the mRNA.
  • Scanning of the mRNA until the start codon is found, usually surrounded by Kozak's sequence.

Elongation

• During elongation, amino acids are added one by one to the preceding amino acid.
• Each addition involves proteins called elongation factors (EFs) and occurs in three steps:
  • Codon recognition: The appropriate tRNA binds to the codon in the A site.
  • Peptide bond formation: Peptidyl transferase (a ribozyme) catalyzes the formation of a peptide bond between the amino acid in the A site and the growing polypeptide chain in the P site.
  • Translocation: The ribosome translocates the tRNA in the A site to the P site, and the tRNA in the P site to the E site, using energy from GTP hydrolysis.

Peptide Bond Formation

• A peptide bond links the carboxyl end of one amino acid with the amino end of another, expelling one water molecule.

Translocation

• The ribosome moves along the mRNA, bringing the next codon into the A site.
• This process involves:
  • Codon recognition: EF-Tu assists in binding the correct tRNA to the A site, using GTP.
  • Translocation: EF-G promotes the movement of the ribosome, using GTP hydrolysis.

Termination

• Termination occurs when the ribosome reaches one of the three stop codons: UAA, UAG, or UGA.
• Release factors recognize the stop codons and trigger the release of the polypeptide chain.
• This process also requires GTP hydrolysis.

Mutations

• Mutations are changes in the nucleotide sequence of a genome.
• Spontaneous mutations occur as a result of natural reactions, such as errors during DNA replication, recombination, or repair.
• Induced mutations occur as a result of exposure to chemical or physical agents (mutagens).

Point Mutations

• Point mutations are changes to single bases.
• These mutations can be divided into two general categories:
  • Base-pair substitutions
  • Base-pair insertions or deletions
• Types of point mutations:
  • Missense: results in a change of a codon, leading to a different amino acid.
  • Nonsense: results in a stop codon, prematurely terminating translation.
  • Sense: results in a stop codon being changed into a “sense” codon.
  • Silent: change in a codon does not result in a change in an amino acid.
• Insertion and deletion mutations:
  • Frameshift: results in a changing of many codons due to the insertion or deletion of bases that are not multiples of three.

Examples and Consequences of Mutations

• A DNA point mutation can lead to a different amino acid sequence and alter protein function, potentially affecting the phenotype.
• Example: A mutation in the DNA sequence can cause a change from glutamic acid to valine, leading to sickle cell anemia.

Levels of Protein Structure

• Primary structure: the sequence of amino acids.
• Secondary structure: local folding patterns such as alpha helices and beta-pleated sheets.
• Tertiary structure: the overall three-dimensional shape of the protein.
• Quaternary structure: the arrangement of multiple polypeptide chains in a multi-subunit protein.

Mutation Examples and Definitions

• Insertion: Addition of nucleotides, which can disrupt the reading frame.
  • Example: Original sequence: ATA ACC GAT CAT GTA; Mutant sequence: ATA ACC GAT CGA TGT AAT
• Deletion: Removal of nucleotides, which can also disrupt the reading frame.
  • Example: Original sequence: ATA ACC GAT CAT GTA; Mutant sequence: ATA ACC GTC ATG
• Frameshift mutations: Involve a number of bases (other than multiples of 3) added or deleted from the DNA. Deletions and insertions are major causes of genetic disorders.
• Point Mutations
  • Silent: has no effect on the protein sequence
    • Original: AGC GTACCC TAC (Ser Val Pro Tyr)
    • Mutation: AGC GTTCCC TAC (Ser Val Pro Tyr)
  • Missense: results in an amino acid substitution
    • Original: AGC GTACCC TAC (Ser Val Pro Tyr)
    • Mutation: AGC GTAAC CTA C (Ser Val Thr Tyr)
  • Nonsense: substitutes a stop codon for an amino acid
    • Original: AGC GTACCC TAC (Ser Val Pro Tyr)
    • Mutation: AGC GTACCCTAG (Ser Val Pro Stop)
• Frameshift Mutations
  • Insertions or deletions of nucleotides may result in a shift in the reading frame or insertion of a stop codon.
    • Original: AGC GT ACCCTAC (Ser Val Pro Tyr)
    • Mutation: AGC GCC C TA (Ser Val Leu Leu)

Mutation Scenarios and Outcomes

• Nucleotide-pair substitution
  • Silent (no effect on amino acid sequence)
  • Missense
  • Nonsense
• Nucleotide-pair insertion or deletion
  • Frameshift causing immediate nonsense (1 nucleotide-pair insertion)
    • Codes: 5' - A U G A A G U U U G G U U A A - 3'
  • Frameshift causing extensive missense (1 nucleotide-pair deletion)
    • Codes: 5' - A U G U A A G U U U G G C U A A - 3'
  • No frameshift, but one amino acid missing (3 nucleotide-pair deletion)
    • Codes: 5' - A U G U U U G G C U A A - 3'

Effects on Protein Product and Phenotype

• Mutations in genes like the CFTR gene can lead to various effects on the protein product and subsequently affect the phenotype.
• Types of mutations in this context include:
  • Missense mutations
  • Nonsense mutations
  • Frameshifts
  • Deletions
  • Splicing mutations