Microbial Genetics: DNA Replication, Gene Expression, and Mutations

Posted on Jan 23, 2025 in Biology

Microbial Genetics

DNA contains thymine, while RNA contains uracil.

DNA is the genetic material in all cellular organisms. Its structure includes:

• The nucleotide, the building block of DNA, consisting of:
  • Phosphate
  • Deoxyribose sugar
  • Nitrogenous base
    • Pyrimidine
    • Purine

Genetics is the study of inheritance in living things, how traits are passed on.

• Cells must be able to self-replicate.
• Information must be duplicated and transmitted to each daughter cell.
• Levels and structure:
  • Genome: The entire cell’s genetic information, all the DNA.
    • Varies in size.
    • Eukaryotic genomes can be similar in size to prokaryotic genomes.
      • The smallest eukaryotic genome can be the size of the largest prokaryotic genome.
  • Chromosome: A single piece of DNA.
    • Can be in an organelle.
    • Cells may have one or many.
    • Contains many genes.
    • Also can be in plasmids or nucleocapsids.
  • Gene: Encodes a protein or RNA, leading to the production of something.
• A cell’s DNA is more than 1000x longer than the cell.
  • Needs packaging – chromatin to prevent tangling.
• Eukaryotic chromosomes
  • Multiple linear strands of DNA
    • Tightly wound around histone proteins.
    • Located in the nucleus.
    • Vary in number.
    • Can exist as diploid or haploid.
• Bacterial chromosomes
  • Single, circular chromosomes.
  • Condensed on histone-like proteins.

• Gene: A segment of DNA that contains the code to make a protein or RNA molecule.
  • Some encode proteins:
    • Structural genes: Encode proteins that perform functions.
    • Regulatory genes: Encode proteins that control gene expression.
      • Others encode RNA that has a role as RNA.
    • Genes for non-coding RNA, involved in the expression of structural genes.
  • Genotype: The collection of all types of genes an organism has. Everything an organism could do based on its genes.
  • Phenotype: The expression of the genotype – the traits that are actually observed.

• DNA replication: Preserving the code and passing it on.
  • Duplication of the genetic code and passage to each offspring.
    • Must be completed during a single generation time.
  • Requires 30 different enzyme actions.
    • Uncoil chromatin.
    • Separate the strands.
    • Copy the template.
    • Ensure no mistakes.
    • Produce 2 new daughter molecules.
• Semiconservative replication
  • Benefits of 2 complementary antiparallel strands:
    • Each strand acts as a template.
    • One old strand pairs with a new one.
    • Allows accurate replication.

Replication starts at the origin.

• Rich in A and T.
  • Why? 2 H bonds vs 3 for G/C.
  • Less energy required to separate strands.
• Generates replication forks:
  • Y-shaped regions where new DNA strands are elongating.
  • Helicases: Enzymes that untwist the double helix at the replication forks.
  • Single-strand binding proteins: Bind to and stabilize single-stranded DNA.
  • Topoisomerase: Relieves “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands.

DNA Polymerase III

• Needs DNA unwound and strands separated first.
• Can add nucleotides to an existing chain.
• Can only add nucleotides in one direction.

DNA replication: Elongation

• The antiparallel structure of the double helix affects replication.
• DNA polymerases add nucleotides only to the free 3′ end of a growing strand.

• • New DNA strand can elongate only in the 5′ to 3′ direction.

• Replication has to happen on both strands at the same time.
• On one template strand of DNA, the DNA polymerase synthesizes a leading strand.
  • • Continuously moving toward the replication fork.
  • On the other new strand, the lagging strand, DNA polymerase must work in the direction away from the replication fork.
    • Synthesized as a series of segments called Okazaki fragments by DNA Pol III.
    • DNA Pol I removes RNA primers and replaces them with DNA.
    • Okazaki fragments are joined together by DNA ligase.

Replication Errors and their detection

• Since they are moving fast, they make mistakes.

The central dogma of molecular biology:

• Central dogma: information starts at DNA, gets converted into RNA, and then into protein.
• Storing information as DNA is not useful.
  • Information must be used.
  • Synthesize an RNA molecule.
    • Transcription – same language, different font. Photocopying, taking pictures.
    • RNA polymerase.
  • Information in the RNA must be acted on.
    • Used to produce proteins.
    • Translation: different language.
    • Ribosomes.
  • Exceptions:
    • COVID, retroviruses.

Synthesis of an RNA transcript

• Stages of transcription:
  • Initiation:
    • RNA synthesis – RNA polymerase (RNAP)
      • It binds to DNA at a sequence called the promoter.
        • If you want a lot of gene product, add more polymerases to the gene.
      • Determined by the strength of binding to the promoter.
      • Needs the help of an activator if the promoter is a poor match to the consensus.
    • RNA denatures DNA strands apart.
    • Assembles RNA.
  • Elongation:
    • RNA polymerase:
      • Unwinds DNA as it moves forward.
      • Adds nucleotides to the 3’ end of the RNA already assembled.
        • RNA strand is complementary to the DNA template strand, with an antiparallel orientation.
  • Termination:
    • Bacteria: special sequences
      • Rho – binds rut – kicks RNAP off RNA.
      • New RNA folds into a hairpin – kicks RNAP off.
    • Eukaryotes: the termination method is unclear.

Major RNAs in gene expression:

1. mRNA:
   1. Transcript of structural genes in the DNA.
   2. Message later read – a series of codons.
      1. 3 nucleotide chunks read/bound by tRNA.
2. tRNA:
   1. Transfer RNA, an adapter that allows the ribosome to read nucleotide language and turn it into protein language.
   2. Adapter between RNA/DNA and proteins.
   3. Anticodon – binds to the codon on mRNA.
   4. 3’ end – binds amino acids.
   5. At least 1 per amino acid.

3. rRNA:

• Ribosomal RNA.
• Major component of ribosomes.
  • Large (acts as an enzyme) and small (used for relatedness) subunits.
• Special sites (A, P, and E) that interact with tRNA.

4. Noncoding RNAs

• May regulate transcription at different levels.

Genetic regulation of protein synthesis and metabolism

• Only want genes when products are required.
• Functional genes are often encoded together in an operon.
• Includes:
  • Structural genes: Encode enzymes/translated from a single mRNA.
  • Promoter: Where RNA polymerase binds.
  • Operator: Where all the other regulators bind, next to the promoter.
  • Regulatory genes: Encode a regulator.
• 2 types of operons:
  • Inducible: Catabolic enzymes. When food is present, make enzymes to break down the food.
    • Default is off – food is available = turns on.
  • Repressible: Anabolic enzymes. Build amino acids, cell wall proteins, DNA, RNA, etc.
    • Building one. Raw materials – things cells need.
    • Default is on.
    • Feedback inhibition – turn the operon off when there is enough.
• The lac operon:
  • Inducible operon: turned on in the presence of lactose (inducer).
    • 3 structural genes.
  • Repressor (LacI) binds the operator – prevents transcription when there is no lactose.
• Repressible operons:
  • Biosynthetic pathways.
    • Default setting → “on”.
    • Turned “off” in the presence of the product of the pathway.
      • Either made enough or getting it from food.
      • Don’t want to make extra.
      • Feedback inhibition.
    • Repressor is only active when bound to the product (a corepressor).
    • Excess nutrient → co-repressor.
      • Blocks the action of the operon.
    • When [nutrient] ↓ → operon de-repressed.

The triplet code:

• Genetic information flows:
  • DNA → RNA → protein.
• Codons: 3 consecutive bases in mRNA.
  • Recognized by anticodons
    • In tRNA.
  • Each calls for a different amino acid to be added to the growing protein.
    • At the ribosome.

• Determines the order of amino acids in a protein (primary structure).
  • 1º structure →→ 3D structure.
    • Protein structure → function.
    • Function → phenotype.
• All life → same code:
  • Genes transplanted from one species to another are still functional.
• More anticodons than amino acids.
  • Some amino acids have more than one codon.
  • Not all mistakes → problems.

Structural features of the Ribosome:

• Made of a mixture of RNA and proteins.
  • The active site is exclusively RNA.
    • RNA is the enzyme that does the work.
  • 2 subunits:
    • Large (2 RNA and ~50 proteins)
      • Attaches amino acids together.
    • Small (1 RNA and ~30 proteins)
      • Interacts with (t and m)RNA – ensures correct codon/anticodon match.
• Three sites for tRNAs:
  • A (aminoacyl) site:
    • “Accepts” amino acids.
    • tRNA enters in successive steps of the elongation cycle.
  • P (peptidyl) site: “Protein”.
  • E (exit) site.

Initiation of Translation:

• mRNA molecule:
  • Leaves the DNA transcription site
    • → ribosomes.
• Ribosomal subunits → special sites for holding mRNA + tRNAs.
  • The small subunit binds mRNA.
  • Then the large subunit binds the small subunit.
    • Helped by initiation factors.
  • Synthesis starts at the first start codon
    • AUG.

Elongation of the Polypeptide chain:

• Amino acids are added one by one.
  • To the C-terminus of the growing chain.
    • Requires: elongation factors.
• Translation moves along mRNA from 5′ → 3′.
  • To make protein N-terminus → C-terminus.

Termination of translation

• Induced by the presence of a stop codon: UAA, UAG, and UGA.
  • aka Nonsense codons.
  • No tRNA with a complementary anticodon.
  • Instead, release factors bind.
    • Break the bond between the final tRNA and the polypeptide chain.
    • Release the peptide from the ribosome.

Polyribosome and “Christmas tree” transcripts

• Ribosomes can add 12-17 amino acids/sec.
• Sometimes need to be faster.
  • Need to change protein levels to respond to new conditions.
• Cells multi-task:
  • Bacteria → multiple RNAP on each gene AND multiple ribosomes.
    • Translate mRNA while it is still being transcribed.
  • Eukaryotes do transcription and translation in different places in the cell.
    • Still do many RNAP/gene.
    • AND many ribosomes/mRNA.
    • Just do them separately:
      • Transcription in the nucleus.
      • Translation in the cytoplasm.

Mutations: Changes in the Genetic Code

• Genetic change → driving force of evolution.
• Mutation: A change in the nucleotide sequence of the genome of a cell or virus.
  • Permanent, heritable.
    • Some phenotypic changes are temporary.
      • Use different genes in different situations.
      • Situations change again, phenotype changes, not necessarily passed on.
      • Change in how a gene is regulated.
  • Mutations are permanent.
• Point mutations: Changes in just one base pair of a gene.
  • Can be divided into two general categories:
    • Nucleotide-pair substitutions.
    • One or more nucleotide-pair insertions or deletions.
• A single nucleotide change in the DNA template can lead to the production of an abnormal protein.
  • A → T transversion leads to Glutamate → Valine at position 6, leading to Sickle Cell Anemia.
• Wild-type: An organism with a natural, non-mutated characteristic.
• Mutant strain: A microorganism with a mutation.
  • Tested for by replica plating or with selective media.
• Sometimes we want to know if conditions/chemicals can lead to mutations.
  • Ames Test
    • Rapid screening system.
      • Detects chemicals with carcinogenic potential.
      • Idea: Any chemical capable of mutating bacterial DNA may be able to similarly mutate mammalian DNA.
        • Bacterial DNA may be easier to mutate – less protected, so can be overly cautious.
      • Take a salmonella strain that needs histidine to survive.
        • His auxotroph.
      • Grow on a medium lacking histidine and look for survivors.
    • Mutations are permanent and heritable.
      • Most with a phenotype are harmful.
        • Many have no visible phenotype.
        • DNA not encoding proteins, multiple codons for the same amino acid, similar codons often encode similar amino acids.

• Some provide adaptive advantages.
  • Those are the ones seen in the Ames Test.

Mutations can be:

• Harmful.
• Neutral.
• Beneficial.
• Spontaneous
  • Random change in the DNA.
    • Sometimes bases get changed as part of the cell’s life.
  • Result of errors in replication.
    • Polymerases proofread, but are not perfect.
• Induced
  • Results of exposure to known mutagens.
    • Like in the Ames Test.
  • Can be targeted or random.
  • Not always due to lab experiments.
    • Exposure to chemicals/radiation as part of life can do this.
    • Free radicals made during metabolism as well.

Categories of mutations:

1. Silent mutation
   1. Nucleotide changes in a gene without changing the amino acid encoded in the final protein.
      1. Multiple codons for the same amino acid.
   2. No effect → silent.
2. Missense mutation
   1. Any change → codon for a different amino acid.
   2. May or may not have an effect on function.
3. Nonsense mutation
   1. Any change → normal codon → stop codon.
4. Frameshift mutations
   1. Addition/deletion of bases in a non-multiple of 3.
      1. Changes the reading frame of the mRNA.
      2. Nearly always → nonfunctional protein.
5. Back-mutation
   1. Mutation reverting a gene to its original base composition.
   2. Fixes an earlier mutation.
   3. Must be a different first mutation for there to be a back mutation.

Repair of mutations:

• DNA polymerases proofread newly made DNA.
  • Feels an incorrect shape when it adds non-matching nucleotides.
  • Chews up the last few bases synthesized.
  • Replaces incorrect nucleotides with new correct bases.
  • Doesn’t get everything.
• Mismatch repair of DNA:
  • Corrects errors in base pairing.
  • Cuts out a region on the new strand.
    • The cell needs to be able to tell the difference between old and new.
  • Replaces it with a better complement to the old strand.
    • Uses the old strand as a template.
  • DNA ligase seals the gaps back together.

DNA recombination:

• Recombination: New DNA → bacteria’s chromosome.
  • Usually from other bacteria.
    • Alive or dead.
  • Also viral integration into the chromosome.
  • End result → a new strain different from both the recipient and the original source of DNA.
• Recombinant organism: Any organism with genes that originated in another organism.
• Gene transfer between organisms → horizontal gene transfer.
  • Genes you didn’t get from your parents.
    • Or the milkman.
    • Or whoever provided the genetic material you were born with.
  • Genes are often exchanged via plasmids.
    • Or fragments of chromosomes.

Plasmid

• • Double-stranded circles of DNA.
    • Usually non-essential.
  • Independently replicating.
    • Though can integrate into the chromosome.
  • Can be transferred from one cell → another.
    • Horizontally – bacterial “sex”.
    • Vertically – passed down to offspring.
  • Contain “special” genes not related to basic life functions.
    • Can confer…
      • Protective traits → such as drug resistance.
      • Production of toxins and enzymes.
    • Can be small or big.
      • ~2-500 kb.
      • Bigger ones may be essential.
        • Gray area with small chromosomes.
  • Important for genetic engineering.
• Genes exchanged usually on plasmids or fragments of chromosomes.
  • Plasmids replicate independently of the bacterial chromosome.
  • Chromosomal fragments must integrate into the host chromosome in order to replicate.

4 ways genes move:

• • Conjugation: Bacterial sex.
    • Involves 2 cell types:
      • F+ and F-.
      • Results in two F+ cells.
    • F+ has a special plasmid and can make a sex pilus.
      • Not all plasmids can do this.
      • Different plasmids → differing levels of promiscuity.
        • Some within the same species (or even strain).
        • Others within the same genus or family.
        • Others share with just about everyone.
    • F+ senses that a neighboring cell is F-.
    • F+ ensnares F- with a pilus.
    • A single strand is transferred to the F- cell.
    • Both cells convert the ssDNA plasmid to dsDNA.
    • The F- cell undergoes gender reassignment surgery → F+.

• • Transformation: Capturing DNA from a solution.
    • Nonspecific uptake of DNA – transformation.
    • Pick up DNA left behind by dead/lysed cells.
      • Same species or different.
      • Can be a different domain.
        • Conjugation/transduction usually requires at least semi-related organisms.
    • Special DNA-binding proteins on the cell wall of certain bacteria.
    • Competent cells – capable of accepting genetic material.
      • Some cells are naturally competent.
        • Sometimes always.
        • Other times only when starving.
      • Some cells we force to be competent.
    • Useful for certain types of recombinant DNA technology.
    • Transfection in eukaryotes.
      • Transformation means immortalizing into a tumor cell.
  • Transduction: Piggyback DNA.
    • Generalized transduction (a virus that infects bacteria).
      • Involves a bacteriophage (a virus that infects bacteria).
      • Mistaken packaging of host genes into a capsule.
        • Generally just packages an X-sized chunk of DNA.
        • Sometimes right-sized degraded host DNA → viral particle.
      • Creates a defective phage.
        • Injects DNA from the last host into a new recipient.
        • Can’t reproduce.
      • DNA carried by the phage is picked up at random – usually.
      • Specialized transducing phages are often insertional phages that pick up DNA next to where they insert into the genome.
        • May result in a partially functional chimeric phage.
  • Transposition: Jumping genes.
    • Gene location isn’t always static.
      • Genetic elements capable of transposition.
        • Moving within a chromosome.
    • Mobile elements called transposons.
    • Move from place to place in the genome.
      • Also onto/off plasmids and viral genomes.
    • Disrupt genes when they land.
    • May mobilize other genes (like antibiotic resistance).

• Genes for resisting antibiotics
  • 1 or many
  • Also to heavy metals or other pollutants
• Genes for synthesizing virulence factors
  • Things that allow it to specifically invade, attach to, or damage host
• Genes for surviving in a new niche
  • Able to grow in stressors at a new site that used to kill it
    • Or kill helpful bacteria that used to fight it
  • Able to eat food present at a new site that it didn’t used to be able to eat
  • Didn’t cause a problem where it lived before but now…

• Pathogenicity islands
• Virulence genes grouped together on a chromosome
  • Large chunks transferred all at once
    • No single genes
    • Dozens to hundreds
    • 10’s to 100’s of thousands of bp
  • Often contain operons or genes of related pathways
  • Not all necessarily genes that cause toxic effects
    • May allow it to use new sugars
      • Allow it to live in a new niche?
    • Antibiotic resistance genes
• Come from other organism(s)
  • Different % G+C than the rest of the host
• Taken up by any mechanism