Pharmaceutical Biotechnology Lecture 9

Level 5, Semester 1 Pharmaceutical Biotechnology, Lecture 9

• Lecture covered microbial genetics, focusing on transcription and translation.
• This lecture discussed the central dogma of molecular genetics, which describes the flow of genetic information from DNA to RNA to protein.
• DNA serves as the template for RNA synthesis (transcription).
• mRNA is a sequence of codons, which are translated into chains of amino acids (polypeptides).
• A codon is a sequence of three nucleotides.

Microbial Genetics

• Lecture summary included transcription and translation.

Protein Synthesis

• DNA → mRNA → Protein
• Transcription
• Translation

Triplet Code

• DNA serves as a template for RNA synthesis.
• A codon is a sequence of three nucleotides that codes for an amino acid.
• Codons are translated into chains of amino acids (polypeptides).

The Genetic Code

• mRNAs consist of codons that code for amino acids.
• The Genetic Code contains 64 codons (4³) corresponding to 20 amino acids.
• An amino acid can be coded for by more than one codon.

Stop Codons

• Three stop codons exist (UAA, UAG, UGA).
• They signal the end of translation.
• AUG is the start codon, coding for methionine.

Transcription

• One strand of DNA (template strand, 3' to 5') is used to create a complementary mRNA strand (5' to 3').
• The other strand of DNA is called the sense strand and is complementary to the mRNA strand (except T is replaced by U).
• Transcription begins at a promoter region (with a TATA box) and ends at the terminator site
• Components of Transcription:
  • Promoter
  • RNA Polymerase
  • Termination Site
• The process of mRNA production from a DNA template.

Transcription in Eukaryotes

• RNA splicing involves removing introns (noncoding sequences) and joining exons (coding sequences)
• A cap and tail are added to the mRNA to facilitate release from the nucleus, stabilize the mRNA, and aid ribosome binding.
  • Cap: single G nucleotide
  • Tail: 50-250 A nucleotides.

Translation

• mRNA translation begins at the start codon (AUG) and terminates at a stop codon (UAA, UAG, or UGA).
• Translation involves three main steps:
  • Initiation
  • Elongation
  • Termination

Initiation (Translation)

• mRNA binds to the small ribosomal subunit.
• Initiator tRNA binds to the start codon (AUG).
• Initiator tRNA carries the amino acid methionine (Met).
• Large ribosomal subunit joins the small subunit.
• Initiator tRNA fits into the P site of the ribosome.
• The other tRNA-binding site (A site) is empty, awaiting the next amino acid-carrying tRNA.

Elongation (Translation)

• New tRNA carrying an amino acid binds to the A site on the ribosome.
• Peptide bond formation between the amino acid at the P site and the amino acid at the A site, releasing the previous tRNA from the P site.
• The ribosome moves along the mRNA
• The P site tRNA leaves the ribosome and the ribosome translocates the tRNA in the A site to the P site.

Termination (Translation)

• A stop codon reaches the ribosome's A site.
• Stop codons do not code for amino acids; they signal the end of translation.
• The completed polypeptide is released from the last tRNA.
• The ribosome subunits separate.

Transfer of Genetic Elements

• Transformation: DNA uptake from the environment.
• Conjugation: DNA transfer between bacteria (often through a pilus).
• Transduction: DNA transfer mediated by a bacteriophage.

Transformation

• Genes are transferred as "naked" DNA from one bacterium to another.
• Frederick Griffith (1928)
• Recipient cell becomes competent by binding special proteins.

Griffith's Experiment

• Used two strains of Streptococcus pneumoniae (one with a capsule, one without).
• Demonstrated genetic material transfer, key step in understanding bacterial transformation.

1- Transformation Process

• A fragment of donor DNA attaches to a competent recipient cell
• Internal DNA processes bind the DNA for replication.

Competence

• A cell able to absorb and utilize foreign DNA from the environment.

Natural Competence

• Special proteins like RecA facilitate DNA uptake in some bacteria.
• Examples include Streptococcus pneumoniae, Bacillus subtilis, Hemophilus influenza, and Neisseria gonorrhoeae.

Artificial Competence

• Induced competence through high concentrations of calcium ions, creating pores in cell membranes.

Conjugation

• Donor bacteria transfers DNA plasmid (usually) to a recipient bacterium via a sex pilus.
• Donor cell must have the F+ factor (plasmid).

Transduction

• DNA transfer between bacteria mediated by a bacteriophage (virus).
• Generalized transduction: packaging of host DNA instead of viral DNA.
• Generalized transduction can lead to recombination with the recipient cells DNA.

Importance of Conjugation

• Allows bacteria to acquire new genes and genetic traits.
• Important in the transfer of antibiotic resistance.

Importance of studying fungi

• Pathogenicity: some fungi cause human diseases.
• Toxin production: mycotoxins harm humans.
• Metabolic activity: damages food and structures.
• Plant diseases: fungi cause many plant diseases.
• Food and pharmaceutical applications: used in food and medicine.

Fungi

• Heterotrophic eukaryotes
• Cell walls contain chitin, mannan, and/or glucan.
• Key types:
  • Yeasts
  • Molds
  • Dimorphic

Fungal Morphology

• Yeasts: Unicellular, oval/spherical cells
• Molds: Filamentous mycelium, hyphae
• Dimorphic: Mold or yeast forms depending on conditions

Fungal Reproduction

• Asexual:
  • Conidiospores
  • Sporangiospores
  • Arthrospores
  • Chlamydospores
  • Blastospores
• Sexual:
  • Zygospores
  • Basidiospores
  • Ascospores

DNA Replication

• Semi-conservative model (Watson & Crick): DNA molecules consist of one parental strand and one new strand.
• Locations: starts at replication bubbles (single origin in prokaryotes, multiple in eukaryotes).
• Enzyme Overview:
  • DNA gyrase (a topoisomerase): Relaxes supercoiled DNA
  • DNA helicase: separates the 2 DNA strands.
  • Single-Strand Binding proteins (SSBs): Keeps separated DNA strands apart
  • Primase: synthesizes short RNA primer, allowing DNA polymerase to start replication.
  • DNA polymerase III: adds nucleotides in the 5' to 3' direction. It is termed "proofreading" or "error correction", as it removes mismatched base pairs.
  • The leading strand (5’ to 3’) is synthesized continuously from the replication fork.
  • The lagging strand (3’ to 5’) is synthesized discontinuously in short segments called Okazaki fragments. Gaps filled by DNA polymerase I and sealed via DNA ligase.

RNA (Structure and Types)

• RNA is a single-stranded polynucleotide.
• RNA contains ribose as the sugar moiety.
• RNA contains Uracil (U) instead of Thymine (T).
• Types Include:
  • mRNA
  • tRNA
  • rRNA

Differences between DNA and RNA

• DNA: double helix; deoxyribose sugar A-T and G-C base pairs
• RNA: single-stranded; ribose sugar, U replaces T in hydrogen bond pairs, migrates out of the nucleus.