Biology Chapter: Translation Mechanism

Peptide Bond Formation

• Peptide bonds form during translocation from the A to the P site on the ribosome.

Termination of Translation

• Occurs when the ribosome encounters a stop codon.
• Stop codons are UAA, UAG, and UGA.
• When a stop codon is encountered, a release factor binds to the A site.
• A water molecule is added to the carboxyl terminus of the nascent polypeptide, causing termination.

The Genetic Code

• Degenerate: More than one codon can specify the same amino acid.
• Ordered: Codons that specify the same amino acids are often similar.
• Two types of degeneracy
  • Partial: The third base can be either two pyrimidines (U or C) or two purines (A or G). Changing the third base from purine to pyrimidine or vice versa will change the amino acid specified by the codon.
  • Complete: Any of the four bases may be present at the third position in the codon and the codon will still specify the same amino acid.

Wobble Base Pairing

• Base pairing between the anticodon of tRNA and the codon of mRNA follows strict base pairing rules for only the first two bases of the codon.
• The base pairing involving the third base of the codon is less stringent, allowing wobble base pairing.
• Mutations at the third position have less impact than the first two positions.
• Amino acids with similar physical properties often share similar codon sequences.
• This minimizes the effect of mutations on protein sequence, structure, and function.
• Some tRNAs contain the base inosine.
• Inosine is produced by a post-transcriptional modification of adenosine.
• The wobble hypothesis predicts that when inosine is present at the 5' end of an anticodon, it will base pair with cytosine, uracil, or adenine codons.

Mutations

• Source of all genetic variation.
• Refer to a change in genetic material or the process by which the change occurs.
• Types:
  • Changes in chromosome number and structure.
  • Point mutations: Changes at specific sites in a gene (substitution, insertion, or deletion).
• A mutant is an organism that exhibits a novel phenotype.
• Heritable changes in genetic material provide the raw material for evolution.
• Recombination mechanisms rearrange genetic variability into new combinations.
• Natural selection preserves the combination best adapted to the existing environment.

Restriction Enzymes

• Cut at specific DNA sequences, usually a specific 6 bp sequence.
• Recognition sequence is palindromic.
• Can make staggered cuts.
• Named after the species in which the enzyme is produced.

Plasmid Vectors

• Plasmids are extrachromosomal DNA molecules that can replicate independently of the host chromosome.
• Features:
  • Multiple cloning site (MCS): Contains unique restriction enzyme sites for inserting foreign DNA.
  • Origin of replication: Allows the plasmid to replicate within the host cell.
  • Selectable marker: Typically an antibiotic resistance gene, allowing for selection of cells containing the plasmid.

Ligations

• The final step in generating recombinant DNA.
• Complementary bases anneal.
• DNA ligase joins the 5' phosphate and 3' OH to reform the phosphodiester bond between adjacent bases (A-T).
• A recombinant DNA molecule is formed following ligation.

Transformations

• A standard tool used in laboratories to propagate recombinant DNA.
• Bacteria like Escherichia coli (E. coli) are used as hosts to maintain and replicate plasmid vectors containing foreign DNA.
• Plasmid vectors need an origin of replication (Ori) to ensure they are copied within bacterial cells.

Gel Electrophoresis

• DNA/RNA is separated by size using agarose gel electrophoresis.
• Smaller fragments travel faster and further.
• Visualization: Stain the gel and visualize under UV light using dyes like ethidium bromide (EtBr).

Protein Separation: SDS-PAGE

• Acrylamide gel electrophoresis (PAGE) is used for protein separation.
• Proteins have a more complex structure and variable charges.
• Solution:
  • Denaturation: Proteins are unfolded into linear forms and given a net negative charge to facilitate separation based on size.
• Method:
  • Reducing agents (β-mercaptoethanol or dithiothreitol) break disulfide bonds.
  • Detergent (SDS) binds to proteins, giving them a uniform negative charge.
  • Heat treatment (at 95°C for 20 minutes) completes denaturation.
• Steps:
  1. Denaturation: Treat proteins with reducing agents, detergent, and heat.
  2. Electrophoresis: Apply current to the acrylamide gel; negatively charged, linear proteins move through the gel; smaller proteins travel faster.
  3. Staining: Stain proteins with bromophenol blue which binds non-specifically to proteins; more protein = more stain binding = darker bands on the gel.

Key Comparisons: Nucleic Acid vs. Protein Electrophoresis

• Nucleic Acids:
  • Linear, negatively charged, separated easily by size in agarose gels.
  • Use fluorescent staining (e.g., EtBr).
• Proteins:
  • Require denaturation and charge equalization with SDS.
  • Separated in acrylamide gels (PAGE).
  • Visualized with protein-specific stains like bromophenol blue.

Third Generation Sequencing

• Long read third-generation sequencing directly sequences the nucleic acid without an amplification step.
• Technologies:
  • Pacific Biosciences Single-Molecule, Real Time (SMRT)
  • Oxford Nanopore technology: Measures 'electrical current intensity' as the nucleic acid passes through a nanopore; no synthesis; resequencing of each piece of library hundreds of times.

Analysis of Sequence Data

• Thousands of reads need to be put back together to give the final sequence.
• Assembly is the process of reconstructing the original sequence.
• Shotgun assembly: Breaking up DNA into smaller fragments and then 'putting it all back together'.

Metagenomics

• An approach to study the genetic material from a mixed population of organisms, often from an environmental sample (e.g., soil, water, or the gut microbiome).
• Allows for the identification of different species within a complex sample.
• Uses either:
  • Amplicon sequencing: Sequencing of a highly conserved gene, like 16S rRNA.
  • Whole-genome metagenomics: Sequencing the entire genome of every organism in the sample.
• It helps to have a reference genome for some of the species in the sample.

Population Genetics:

• Genotype Frequency: The proportion of different genotypes in a population.
• Hardy-Weinberg Principle: Provides a mathematical model to predict allele and genotype frequencies in a population under certain conditions (no mutation, random mating, etc.).
• Hardy-Weinberg Equilibrium: Populations in equilibrium will maintain constant allele frequencies over time unless acted upon by evolutionary forces.
• Simple Measures of Genetic Diversity: Allele and genotype frequencies are used to assess genetic diversity within populations, which is critical for understanding evolution and adaptation.

Evolution:

• Sameness (Homology): Similar structures across species that are conserved over time indicate descent from a common ancestor (e.g., limb structures in various animals).
• Difference (Adaptation): Species exhibit differences based on environmental challenges, with some changes driven by adaptation, while others may arise without adaptive significance.
• Microevolution: Refers to small-scale changes within populations, often related to genetic diversity and allele frequency.
• Macroevolution: Involves large-scale changes, including speciation and divergence over long periods.
• Evidence for Evolution includes fossils, speciation, and homology.
• Speciation: Darwin's finches, for example, show how environmental factors and variation within a species can lead to the formation of new species through adaptive divergence.

Natural Selection

• Explains how organisms accumulate traits suited to their environments.
• Variation and inheritance: Genetic variation is the basis for natural selection, and mutations provide the raw material for evolution.
• Non-random survival and reproduction: While mutations are random, the survival and reproduction of organisms are non-random and shaped by environmental pressures.
• Misconceptions: Natural selection does not act on individuals but on populations. Over many generations, populations evolve to become better adapted to their environments.

Explore the fascinating process of translation in this quiz, focusing on peptide bond formation, the genetic code, and termination of translation. Understand the significance of codons and the concept of wobble base pairing. Test your knowledge on how proteins are synthesized in cells.