Lecture 6 - Microbial Genetics1

Microbial Genetics

Important Definitions

• Genome: All of the DNA present in a cell or virus.

• Genotype: The specific set of genes an organism has.

• Phenotype: The observable characteristics of an organism.

• Central Dogma: Describes the flow of genetic information from DNA to RNA to protein.

DNA as Genetic Material

• Historically, there was significant debate about which molecules were responsible for heredity, with a focus on proteins due to their complexity.

• DNA, perceived as too simple due to having only four nucleotides compared to the twenty amino acids in proteins, was ultimately proven to be the genetic material through experimental evidence.

Flow of Information

• Genetic information flows in two primary directions:

  • DNA replication: Transfer of DNA to a new generation.

  • Gene expression: Within a single cell, involving

    • Transcription: Conversion of DNA to mRNA.

    • Translation: mRNA is translated into protein.

DNA Structure

• DNA consists of four different nucleotides categorized into two types:

  • Pyrimidines: Cytosine and Thymine.

  • Purines: Adenine and Guanine.

• Adenine pairs with Thymine (2 hydrogen bonds) and Guanine pairs with Cytosine (3 hydrogen bonds).

• The structure of DNA includes:

  • A 5’ end with a phosphate group.

  • A 3’ end with a hydroxyl group.

General Characteristics of Genes

• A gene is a segment of DNA coding for a specific protein, tRNA, or rRNA.

• In eukaryotes, genes are split into coding regions (exons) and non-coding regions (introns), with mRNA modified to include only exons.

• Most prokaryotic genes lack introns, which simplifies their gene structure and protein translation processes.

Bacterial DNA

• Bacterial DNA is circular and exhibits coiling and supercoiling due to mechanisms that involve nicking the DNA and applying torsion.

• DNA gyrase, a topoisomerase II enzyme unique to bacteria, plays a critical role in creating supercoils, while topoisomerase I removes excessive supercoiling, and antibiotic treatments often target DNA gyrase to inhibit bacterial growth.

Archaeal DNA

• Similar to eukaryotic chromatin, archaeal DNA employs five types of proteins called histones around which DNA coils to form nucleosomes, protecting it from degradation by nucleases.

Chloroplast Genomes

• Chloroplast genomes are circular and similar to bacterial genomes, encoding genes crucial for photosynthesis, rRNAs, and tRNAs.

• Chloroplasts share similarities with various bacteria, especially noteworthy is their intron content compared to bacterial genomes.

Mitochondrial Genomes

• Mitochondrial genomes, involved in oxidative phosphorylation, tend to have fewer genes than chloroplasts.

• Mitochondrial DNA can exist in various forms, such as circular and linear, yet in humans, it encodes only thirteen proteins.

DNA Replication

• DNA replication is the process that ensures genetic continuity across generations, showing differences in mechanisms between eukaryotes and prokaryotes.

• Models of DNA replication include:

  • Conservative: Parental strands reattach after replication.

  • Semiconservative: Each new strand contains one old and one new DNA strand (the correct model).

  • Dispersive: Each strand is a mix of new and old DNA.

Prokaryotic DNA Replication

• Initiates at a single origin of replication, forming two replication forks that move in opposite directions to duplicate the entire strand.

• Unlike bacterial cells, some archaeal cells might possess multiple origins of replication.

Eukaryotic DNA Replication

• Characterized by multiple origins of replication due to longer, linear chromosomes, allowing more efficient replication of the genome.

Replication Machinery

• Eukaryotes and Archaea share similarities in their DNA replication machinery, which is distinct from that of bacteria. The essential enzyme involved in DNA synthesis is DNA polymerase, working in the 5’ to 3’ direction.

Priming DNA Replication

• Primase synthesizes short RNA primers, allowing DNA polymerase III to initiate DNA synthesis since it cannot start without a primer.

Leading and Lagging Strands

• The leading strand is synthesized continuously, while the lagging strand is created in fragments known as Okazaki fragments, requiring more complex processing.

Processing Lagging Strands

• DNA polymerase I removes RNA primers and replaces them with DNA, while DNA ligase connects the Okazaki fragments.

Proofreading Mechanisms

• DNA replication is subject to errors, which are corrected through proofreading mechanisms by DNA polymerases that can excise incorrectly paired bases based on hydrogen bonding patterns.

Termination of Replication

• The separation of newly synthesized genomes occurs through topoisomerases. Following replication, polymerase I and ligase finalize the gaps to complete the process.

Genes and Genetic Exchange in Prokaryotes

• Prokaryotes reproduce asexually but can undergo horizontal gene transfer, which enhances genetic diversity. Methods include:

  • Transformation: Uptake of DNA from the environment.

  • Transduction: Gene transfer via bacteriophages.

  • Conjugation: DNA transfer through direct cell-to-cell contact.

Rolling Circle Replication

• Involves plasmids during conjugation in bacteria, specifically E. coli, where one strand is nicked to allow replication to initiate.

Gene Structure

• Genes are crucial for cellular function, coding for products such as rRNA, tRNA, and mRNA, which are integral to protein synthesis.

• Prokaryotic genomes generally do not have overlapping genes, contrasting the interruptions seen in eukaryotic genes (introns).

Bacterial Transcription

• Transcription is simpler with fewer steps compared to replication and is performed by bacterial RNA polymerase.

• It involves three main processes: initiation, elongation, and termination, requiring the sigma factor for start initiation.

Archaeal Transcription

• Similar to bacterial transcription with one RNA polymerase, archaeal transcription involves more intricate processes like post-transcriptional modifications due to the presence of introns in some genes.

Translation

• The process of protein synthesis from mRNA, codons of three nucleotides are recognized by tRNA carrying amino acids, with start and stop codons defining functional parameters.

• Ribosomes, made of subunits, are central to this process enabling polypeptide synthesis and efficient translation using polyribosomes.

Translational Elongation

• Three significant sites in ribosomes facilitate elongation: A site (for tRNA entry), P site (for peptide bond formation), and E site (for tRNA exit).

Translational Termination

• Occurs when stop codons occupy the ribosome's A site, leading to a halt in translation and disassembly of the ribosomal complex.