Microbial Genetics

Microbial Genetics

Structure and Function of Genetic Material

  • Genetics: The study of genes, their information-carrying capacity, expression, and replication.

  • Chromosomes: Structures containing DNA that physically carry hereditary information; composed of genes.
      - Genes: Segments of DNA encoding functional products, usually proteins.
      - Genome: The complete set of genetic information in a cell.

The Genetic Code

  • Genetic Code: A set of rules determining how a nucleotide sequence is converted into an amino acid sequence of a protein.

  • Central Dogma of Molecular Biology: Describes the flow of genetic information:
      - Genotype: The genetic makeup of an organism.
      - Phenotype: The observable expression of the genotype, dictated by gene expression.

DNA and Chromosomes

  • Most bacteria possess a single circular chromosome made of DNA and associated proteins.

  • Short Tandem Repeats (STRs): Repeating sequences of non-coding DNA found within the genome.

DNA Replication

  • DNA Structure:
      - Forms a double helix.
      - Backbone: Composed of deoxyribose-phosphate.
      - Base Pairing: Strands of nucleotides are connected by hydrogen bonds between Adenine (A) & Thymine (T) and Cytosine (C) & Guanine (G).
      - The strands are antiparallel (one runs 5' to 3' while the other runs 3' to 5').
      - The sequence of nitrogen-containing bases provides genetic instructions.

Mechanism of DNA Replication

  • One strand serves as a template for the synthesis of a complementary strand.

  • Key Enzymes Involved:
      - Topoisomerase and Gyrase: Relax supercoiling ahead of the replication fork.
      - Helicase: Separates the two strands of DNA, creating a replication fork.

  • DNA Polymerase: Enzyme that adds nucleotides to the growing DNA strand in the 5' to 3' direction, initiated by an RNA primer.
      - Leading Strand: Synthesized continuously.
      - Lagging Strand: Synthesized discontinuously, forming Okazaki fragments.
      - RNA primers are removed by DNA polymerase, and Okazaki fragments are joined by DNA polymerase and DNA ligase.

Summary of Events on the DNA Replication Fork

  • Proteins stabilize unwound parental DNA.

  • Leading Strand Synthesis: Continuous synthesis by DNA polymerase.

  • Lagging Strand Synthesis: Discontinuous, initiated by RNA primer through primase, later replaced with DNA by DNA polymerase and joined by DNA ligase.

Important Enzymes in DNA Replication, Expression, and Repair

  • DNA Gyrase: Relaxes supercoiling ahead of the replication fork.

  • DNA Ligase: Joins DNA strands (e.g., Okazaki fragments, new segments in excision repair).

  • DNA Polymerases: Synthesize DNA, proofread, and facilitate repair.

  • Endonucleases: Cut the DNA backbone for repairs and insertions.

  • Exonucleases: Cut DNA from exposed ends to facilitate repairs.

  • Helicase: Unwinds double-stranded DNA.

  • Methylase: Adds methyl groups to selected bases in newly made DNA.

  • Photolyase: Uses visible light energy to separate UV-induced pyrimidine dimers.

  • Primase: An RNA polymerase that synthesizes RNA primers from a DNA template.

  • Ribozyme: RNA enzyme that removes introns and splices exons together.

  • RNA Polymerase: Copies RNA from a DNA template.

  • snRNP (small nuclear ribonucleoproteins): RNA-protein complex that removes introns and splices exons together.

  • Transposase: Cuts and reseals DNA in transposons.

Adding a Nucleotide to DNA

  • Nucleotide Addition Process:
      - When a nucleoside triphosphate bonds to the sugar during DNA synthesis, it loses two phosphates, providing energy for the reaction through hydrolysis of phosphate bonds.

  • Energy Requirements for Replication:
      - Supplied by nucleotides.
      - Hydrolysis of two phosphate groups on ATP generates energy.

Characteristics of Bacterial DNA Replication

  • Bidirectionality: Most bacterial DNA replication proceeds in two directions.

  • Daughter Cells: Each offspring cell receives one copy of the original DNA molecule.

  • Replication Accuracy: High fidelity due to the proofreading capability of DNA polymerase.

RNA and Protein Synthesis

  • Ribonucleic Acid (RNA):
      - Structure: Single-stranded nucleotide with a 5-carbon ribose sugar.
      - Contains uracil (U) instead of thymine (T).

  • Types of RNA:
      - Ribosomal RNA (rRNA): Integral part of ribosomes.
      - Transfer RNA (tRNA): Transports amino acids during protein synthesis.
      - Messenger RNA (mRNA): Carries coded information from DNA to ribosomes.

Transcription in Prokaryotes

  • Definition: Synthesis of a complementary mRNA strand from a DNA template.

  • Process Initiation: Begins when RNA polymerase binds to the promoter sequence on DNA.

  • Direction of Transcription: Proceeds in the 5' to 3' direction; only one of the two DNA strands is transcribed.

  • Termination: Occurs when RNA polymerase reaches the terminator sequence on DNA.

Translation

  • Definition: mRNA is translated into proteins.

  • Codons: Groups of three mRNA nucleotides that code for a specific amino acid; 61 sense codons exist for 20 amino acids.
      - The genetic code is degenerate, meaning most amino acids are encoded by multiple codons.

  • Translation Process:
      - Starts at AUG (start codon) and ends at nonsense codons (UAA, UAG, UGA).
      - tRNA molecules transport required amino acids to the ribosome.
      - tRNA has an anticodon to base-pair with mRNA codons.
      - Amino acids are linked by peptide bonds.

The Process of Translation Step-by-Step

  • Translation Begins:
      - tRNA carrying the first amino acid pairs with the start codon at the P site on the ribosome.
      - A second tRNA carrying another amino acid approaches and pairs with the second codon at the A site.
      - The first amino acid joins the second via a peptide bond.
      - The ribosome moves along the mRNA, shifting the first tRNA to the E site.
      - This continues, allowing the growing polypeptide chain to elongate.

  • Termination of Translation:
      - Stops when reaching a stop codon, leading to release of the polypeptide. All components disassemble afterward.

Simultaneous Transcription and Translation in Bacteria

  • In bacteria, translation can begin before transcription is complete, allowing for rapid protein synthesis.

Transcription in Eukaryotes

  • Location of Transcription and Translation:
      - Transcription occurs in the nucleus; translation occurs in the cytoplasm.

  • Exons vs Introns:
      - Exons: Regions of DNA that code for proteins.
      - Introns: Non-coding regions removed during RNA processing.

  • Role of snRNPs: Small nuclear ribonucleoproteins (snRNPs) splice exons together after removing introns.

Changes in Genetic Material

  • Mutation: A permanent alteration in the DNA base sequence, which can be neutral, beneficial, or harmful.

  • Mutagens: Agents that induce mutations.

  • Spontaneous Mutations: Occur in the absence of mutagens and arise naturally.

Examples of Mutations

  • Base Substitution: A thymine incorporated opposite guanine by mistake, leading to potential codon changes during transcription and translation.
      - If adenine pairs with the new thymine, an AT pair is formed instead of the original GC pair, impacting amino acid coding during translation.

  • Missense Mutation: Change in one base alters an amino acid in the resulting protein, potentially altering function.

  • Nonsense Mutation: Base substitution introduces a premature stop codon, truncating protein synthesis.

  • Frameshift Mutation: Insertion or deletion of one or more nucleotide pairs shifts the translational reading frame, leading to extensive changes in the resulting polypeptide.

Chemical Mutagens

  • Nitrous Acid: Causes adenine to pair with cytosine instead of thymine.

  • Nucleoside Analogs: Incorporate into DNA instead of normal bases, leading to incorrect base pairing.

Radiation Effects on DNA

  • Ionizing Radiation: Forms ions that oxidize nucleotides and can break the deoxyribose-phosphate backbone.

  • Ultraviolet Radiation: Causes formation of thymine dimers, disrupting base pairing; Photolyases can repair these dimers.

  • Nucleotide Excision Repair: Enzymes cut out incorrect bases and replace them with correct ones, restoring DNA integrity.

Genetic Recombination by Crossing Over

  • DNA from one cell aligns with recipient cell DNA, involving matching base pairs and potential genetic alterations through RecA protein activation leading to strand joining.

Griffith's Experiment on Transformation

  • Transformation: The process where DNA is transferred from one bacterium to another as “naked” DNA, leading to genetic changes in the recipient organism.

Mechanism of Genetic Transformation in Bacteria

  • Steps:
      - Donor DNA enters the recipient cell, aligning with complementary bases.
      - Recombination occurs to incorporate donor DNA into the recipient's chromosome.
      - Any remaining, unrecombined donor DNA is degraded.

Plasmids and Transposons

  • Mobile Genetic Elements: Move between chromosomes or cells, found in both prokaryotic and eukaryotic organisms.

  • Plasmids: Self-replicating circular DNA molecules that often encode traits enhancing bacterial pathogenicity.

  • Transposons: Segments of DNA that can move within the genome, containing insertion sequences that code for transposase enabling movement.

R Factor Plasmids

  • Conjugative Plasmids: Carry genes necessary for conjugation, facilitating horizontal gene transfer among bacteria.

  • Dissimilation Plasmids: Code for enzymes that allow bacteria to metabolize unusual compounds.

  • Resistance Factors (R Factors): Encode for antibiotic resistance.

Conjugation in Bacteria

  • Direct transfer of plasmids between bacteria requiring cell-to-cell contact via sex pili.

  • F Factor: A specific plasmid that designates donor (F+) cells; converts F– cells to F+ upon transfer.

Conjugation in E. coli

  • Hfr Cells: High frequency of recombination cells that have integrated the F factor into their chromosome, allowing for transfer of chromosomal genes during conjugation.

Transposons Overview

  • Transposons (Jumping Genes): Segments that relocate within the genome, affecting gene function and potentially contributing to antibiotic resistance.

  • Insertion Sequences: Contain transposase genes essential for facilitating transposition.

Transduction in Bacteria

  • Mechanism: DNA is transferred from one bacterium to another via a bacteriophage.
      - Generalized Transduction: Involves random packaging of bacterial DNA.
      - Specialized Transduction: Specific bacterial genes are packaged and transferred.

Genes and Evolution

  • Importance of mutations and genetic recombination in creating diversity, which serves as raw material for evolution and is acted upon by natural selection to enhance survival of fitter organisms in their environment.