MIC102 MT2

WQ2025 Sam Munoz Diaz

What is the central dogma of molecular biology?

DNA → RNA → Protein.

Genetic information flows from DNA to RNA (transcription), then from RNA to protein (translation)

What are some of the key enzymes involved in DNA replication? (5)

DNA Polymerase- Adds nucleotides to the growing DNA strand

Helicase: Unwinds the DNA double helix

Primase: Lays down RNA primers for DNA polymerase

Ligase: Seals gaps between Okazaki fragments

Single-strand binding proteins: stabilize unwound DNA

Why does DNA replication require an RNA primer?

DNA polymerase needs a free 3’ hydroxyl group (-OH) to start adding nucleotides

What is the leading vs lagging strand in DNA replication?

Leading strand: Synthesized continuously in the 5’ to 3’ direction

Lagging strand: synthesized in short Okazaki fragments that are later joined by DNA ligase

Where does bacterial DNA replication start and stop?

Starts at the Origin of Replication (OriC): An AT-rich sequence easier to unwind

Stops at the Terminator (Ter): ensures full replication and prevents premature stopping

Why do bacteria replicate DNA faster in rich media?

In nutrient-rich conditions, new rounds of replication begin before the previous one finishes, ensuring rapid cell division

How does DNA mismatch repair work?

• MutS protein detects mismatched bases

• DNA polymerase fixes mistake

• Methylation marks the old (correct) strand as a reference

What is the role of DNA gyrase?

Relieves supercoiling tension during DNA replication. It’s a target for antibiotic since humans don’t have DNA gyrase

What does RNA polymerase do?

Synthesizes RNA using DNA as a template (DNA-dependent RNA polymerase). It doesn’t need a primer like DNA polymerase

What are sigma factors and why are they important?

Help RNA polymerase recognize promoters to start transcription. Different sigma factors regulate genes needed under different conditions (e.g. stress response, nitrogen metabolism)

What is the function of ribosomal RNA (rRNA)?

Forms part of the ribosome, which is the site of protein synthesis. It accounts for 90% of cellular RNA

How does translation work?

1. mRNA provides the code

2. tRNA delivers amino acids based on codon-anticodon matching

3. Ribosome catalyzes peptide bond formation to make proteins

What is the function of tRNA?

tRNA reads mRNA codons and delivers the corresponding amino acid to the ribosome

How do bacteria achieve rapid protein synthesis?

Transcription and translation occur simultaneously (coupled transcription-translation), unlike in eukaryotes

What is the difference between factor-independent and factor-dependent transcription termination?

Factor-independent: RnA forms a hairpin loop, causing detachment

Factor-dependent (Rho dependent): The Rho protein releases RNA polymerase from DNA

What is the difference between mRNA, tRNA, and rRNA?

• mRNA: Messenger RNA; contains instruction for protein synthesis

• tRNA: Transfer RNA; carries amino acids to ribosomes

• rRNA: Ribosomal RNA; structural and enzymatic component of ribosomes

What is an operon?

A cluster of genes transcribed together as a single mRNA, allowing coordinated gene expression (e.g., lac operon for lactose metabolism)

What are the 3 ways for gene expression

transcription: controls whether mRNA is made

translation: controls how much protein is made from mRNA

Post-translation: Controls protein activity after it is made

leaky transcription

bacterial genes are never really off, always a little transcription

Constitutive genes (housekeeping genes)

“always” on and required for basic functions

Regulated genes

can be inducible (turned on when needed) or repressible (turned off when unnecessary)

is lac operon regulated or constitutive

regulated but is leaky and always has lactose permease present

lacZ, lacY, LacI functions

• lacZ makes beta-galactoside and that makes allolactose

• lacY makes lactose permease, which allows lactose to be permeable and move through the membrane

• LacI makes a protein/transcription factor that represses the lac operon, thus lac-i down-regulates the lac operon.

a. Negative control of an inducible gene

• A repressor protein binds to the operator, blocking RNA polymerase and preventing transcription

• When an inducer binds to the repressor, it changes shape and releases from the operator, allowing transcription to occur

• Ex: lac operon

B. Negative control of repressible gene

• the repressor protein (aporepressor) is initially inactive, allowing transcription

• When a corepresor binds to the repressor, it activates it, and the complex binds to the operator, blocking transcription

• ex. the trp operon (trp biosynthesis)

C. Positive control of an inducible gene

• activator protein alone cannot bind to the DNA, so transcription does not occur

• Inducer binds to activator, changes shape and binds to activator-binding site, recruiting RNA polymerase and inducing transcription

• ex. CAP-cAMP system in the lac operon (glucose control)

D. Positive control of a repressible gene

• activator protein is required for transcription

• Inhibitor binds to activator, prevents it from binding to DNA, stopping transcription

• ex. certain metabolic pathways where gene expression stops when end product is abundant

How do bacteria regulate catabolic (breakdown) enzymes?

• If substrate (lactose) is present, enzymes are made (on)

• If a preferred energy source (glucose) is available, bacteria repress unnecessary enzymes (off)

How do bacteria regulate biosynthetic (anabolic) enzymes?

• If end product (trp) is available, enzyme production stops (off)

• If end product is absent, enzymes are synthesized (on)

How does the lac operon work?

• lac operon controls lactose metabolism

• Lacl repressor bind the operator and blocks transcription

• Allolactose (inducer) binds to Lacl, removing it from the operator, allowing transcription

• CAP-cAMP complex enhances transcription but is only active when glucose is low

global regulator in catabolite repression

cyclic AMP receptor protein (CRP or CAP)

How does catabolite repression regulate the lac operon?

• when glucose is present, cell prefers glucose and won’t use lactose

• glucose lowers cAMP and adenylate cyclase (AC) activity, so CAP cannot activate the lac operon (no transcription of catabolic genes)

• When glucose is low/used up = AC activity resumes, cAMP increases, CAP binds DNA, and the lac operon can be transcribed

What is attenuation in the trp operon? (if trp is high?low?)

• If trp is high, ribosomes move quickly, causing terminator loop in mRNA that stops transcription

• If trp is low, ribosome stall, forming an anti-terminator loop, allowing transcription to continue

  • Attenuation: transcription is terminated shortly after initiation before completing the transcript

Regulatory RNAs (sRNA): Where they bind

• 6S RNA made in stationary phase binds to sigma-17 RNAP and prevents genes from being transcribed

• Some bind DNA directly

• Some bind to mRNAs and stop transcription/stability

• Some bind to mRNAs and alter translation

Global regulation: regulon and modulon

• Regulon: one regulator affects each operon in regulon; sigma factors control regulons

• Modulon: one regulator affects multiple regions; modulo’s control sets of regulons

Two component signal transduction systems

• Sensor kinase: Histidine kinase + response regulator; transmembrane protein that can sense environmental signals

• Response regulator: DNA binding and stress responses; targets gene to be controlled

Why is motility highly regulated

• cells need to move to gather nutrients or avoid stresses/dangers

• flagella rotate counterclockwise to move cells cells better (clockwise rotation causes random tumbling)

• chemotaxis

What is chemotaxis and how is it regulated? (CheA, CheY, CheB functions)

• Methyl-accepting chemotaxis proteins (MCPs) sense attractants/repellents; bind signal molecules

• CheA (sensor kinase): senses MCP conformation

• CheY: response realtor, controls flagella rotation direction

• CheB: another response regulator, methyl transferase

What happens when an attractant is not sensed (CheA, CheY, CheZ)

• CheA autophosphorylates and passes the signal to CheY RR

• CheY-P makes flagella turn clockwise (tumbles)

• CheZ phosphatase dephosphorylates CheY (stops tumbling)

• Alternates tumbles and runs

What happens when an attractant (i.e. glucose) is sensed (CheA, CheY, CheZ?)

• Attractant binds to MCP, inhibiting CheA autophosphorylation

• No active CheA means no active CheY causing counterclockwise flagella motion (run)

• Methylations of MCP resets conformation, allowing CheA to activate again, requiring higher attractant levels to inhibit it again

• accommodation ensures bacteria moves towards increasing attractant concentration

What happens when repellents are sensed?

• repellent binds to MCP, stimulating CheA autophosphorylation (tumbles)

• high activity Che A phosphorylates CheB, removing methyl groups from MCP (sense smaller quantities of repellent)

What is quorum sensing, and what is it used for?

• Bacteria communicate using autoinducers like homoserine lactones (AHLs) in gram-negatives and specific peptides in gram-positives

• Cells wait until population density is high before activating genes

• Used for virulence, biofilm formation, light production

How does Luxl-LuxR quorum sensing system work?

• Luxl produces AHL autoinders

• At high AHL levels, it binds LuxR, activating lux operon

• Leads to luciferase production, making bacteria glow

What are the 3 requirements for natural selection?

1. variation

2. heritability

3. Differential reproductive success

Effects of mutations

• Neutral: no effect (due to redundancy in genetic code)

• Harmful: lose function

• Beneficial: Rare but can create new functions that help survival

What are spontaneous vs induced mutations?

• Spontaneous mutations happen during DNA replication (error rate is low due to proofreading)

• Induced mutations are caused by external factors (mutagens) like chemicals or UV radiation

What are point mutations and their effects? (3)

• silent mutation: no amino acid change

• Missense mutation: one amino acid changes, possible altering function

• Nonsense mutation: creates a premature stop codon, stopping protein production

What are frameshift mutations?

Insertions or deletions shift the reading frame of codons, altering every amino acid after the mutation. Often leads to nonfunctional proteins

Mutation definition

any change in DNA sequence inherited by offspring; some have no observable affect on phenotype

Why do bacteria have mutations if DNA polymerase is so accurate?

While error rate is low (1 in a billion base pairs), bacteria reproduce in huge numbers

What are auxotrophs?

• Bacteria that lose ability to synthesize a building block or other growth factor.

• Are temperature sensitive (TS) mutants and conditional lethal (cannot grow in high temps)

Enrichment (auxotroph screening)

grow in a medium without the component you want to find auxotrophs for

• Add penicillin to kill non-auxotrophs

Screening (studying mutants)

plate onto permissive medium

• take colonies from master plate and restreak them onto medium without component (colonies that can’t grow on new medium are auxotrophs)

Mobile elements (transposable elements)

• pieces of DNA that can move around a chromosome

• Non-composite transposon: contain drug resistance gene

• Composite transposon: two sets and repeats surrounding other genes

Simple vs Replicative Transposition

Simple: Cut and paste TE into target DNA

Replicative: Copy and paste Te into both target DNA and new cell

What is site-directed mutagenesis and how does it work?

It is a targeted mutation method using PCR with designed primers to introduce specific mutations into a gene, which is then incorporated by homologous recombination

What is complementation and how does it confirm a mutant gene’s effect?

Complementation tests whether a wild-type (WT) gene restores the normal phenotype. If it does, the mutant gene is confirmed to cause the observed effect

What is horizontal gene transfer (HGT)?

Genetic exchange in prokaryotes that doesn’t rely on reproduction. Small amounts of DNA exchanged

1. Transformation

2. Transduction

3. Conjugation

How is DNA taken up and integrated during natural transformation?

1. Competence proteins bind dsDNA

2. one strand is degraded, the other enters the cell

3. RecA coats ssDNA, finds a homologous sequence, and facilitates recombination into the chromosome

What are some artificial transformation techniques to introduce DNA into the cell?

• Heat shock, electroporation, firing DNA coated into a metal into the cell

• Typically uses plasmid DNA

What is transduction?

The transfer of genetic material from one organism to another by a virus.

Generalized Transduction

• Process where a phage accidentally packages random bacterial DNA into its capsid instead of viral DNA

• During rolling-circle replication, phages use pac sites to package viral genomes but bacterial DNA with similar sites can also be mistakenly packaged (phage P22)

Rolling circle replication

• important to phage replication and bacterial conjugation

• circular DNA is nicked and unwound allowing polymerase to use exposed 3’ strand to begin synthesis allowing replication to continue around entire molecule if small and minimal supercoiling

What is specialized transduction and which phage is the model for it?

Occurs when a temperate phage (λ phage) integrates into the host genome at a specific attachment site (att) and remains dormant until later point (lysogeny)

How does specialized transduction occur in λ phage?

λ phage integrates into attB sites in E. coli, near gal and bio genes. Abnormal excision can mistakenly include one of these genes in the phage genome.

How does the transferred bacterial DNA recombine in specialized transduction?

The phage with bacterial DNA can use homologous recombination to integrate into a new host genome, depending on the number of crossover events.

What is bacterial conjugation and what is the key plasmid involved?

Direct transfer of plasmids between bacterial cells; F-plasmid

What structures does F-plasmid encode for conjugation?

Encodes a sex pilus to bring cells together and may encode Type IV secretion system to transfer DNA

How is DNA transferred during conjugation?

Tpe IV recreation system transfers plasmid DNA after it is nicked at the brit and processed by rolling circle replication

What is the F plasmid's role in bacterial conjugation?

• Facilitates conjugation and can carry insertion sequences, promoting recombination with the bacterial chromosome to form Hfr strains.

• It can transfer integrated plasmids, often bringing host genome segments, and the transfer duration helps map gene positions

Potential applications for cloning

• express a gene for study

• express a gene in a different organism for beneficial effect

• Get gene into a system for mutagenesis

• keep gene in a plasmid for stable replication

Polymerase Chain Reaction (PCR)

Generates copies of specific DNA sequences in an exponential manner. Each added cycle double the number of copies of the fragment

3 Steps to PCR

1. Denaturing: heat briefly to separate DNA strands

2. Annealing: cool to allow primers to hydrogen bond with ends of target sequence

3. Extension: DNA polymerase adds nucleotides to 3’ end of each primer

Restriction Enzymes

• protect bacterial DNA by cutting unmethylated foreign DNA

• Cut DNA exposes ends, targeting them for degradation

• Used for DNA cloning

What is CRISPR-Cas, and how does it function?

• bacteria store viral DNA in CRISPR arrays as memory of infections

• Cas proteins use stored sequences to recognize and cut invading DNA

• Can be used for genome editing

Genome

Organism’s complete collection of heritable information stored in DNA (all of an organism’s DNA)

Genomics

studying the entire genome of an organism

DNA sequencing

• Take fragment you want to sequence, add in DNA polymerase and 4 dNTPs. Add radioactive ddNTP in 1 of 4 reactions. ddNTP will stop reaction. (Sanger method)

• Good for short sequences but has a variety of problems

Automated Capillary Sequencing

If the labels are fluorescent instead of radioactive, you can run all reactions in one lane.

Downsides to Sanger-style sequencing

• long reads but slow and not always accurate

• Requires you to fragment genomic DNA and clone into plasmids or bacterial artificial chromosomes (BACs) if you want to sequence a whole genome

• Each plasmid has to be sequenced separately

Sequencing by synthesis

uses polymerases and enzymes to create copies of the DNA

Nanopore sequencing

DNA goes through machine reading the electrical current from H bond donors and acceptors

What is genome annotation?

Process of predicting features in a DNA sequence, such as genes, operons, promoter sequences

How is a genome annotation performed?

• compare predicted gene sequences to databases of known genes

• Identify homologs

• find matches to known protein domains

What are the parts to a pangenome?

• Core genome: sequences shared between all individuals of the species

• Accessory or dispensable genome: partially shared and strain-specific genes (sometimes in plasmids)

Functional Genomics

Diagram of all the cell’s genes products to determine what it can and can’t do

What is metagenomics?

The study of many entire genomes in total

• genomics and metagenomics good to study bacteria that can’t be grown in a lab or mixed samples from any environmental source

List 3 applications of genome sequencing

agriculture, energy, disease

Transcriptome

Organism’s complete collection of RNA transcripts

Proteome

organism’s complete collection of proteins. Proteins change according to time, cell type, and cell state

• Note: if transcriptomes are changing so are proteomes

  • genome → transcriptomes → proteome

Metabolome

Organism’s complete collection og metabolites

• Can’t get all metabolites with a single method; metabolite is usually defined as any molecule less than 1kDa in size

Order of data to find biological phenotype

Genomics (DNA) → Transcriptomics (RNA) → Proteomics (Proteins) → Metabolomics (biochemical) → Biological Phenotype

Why is morphology not always enough to categorize prokaryotes?

• uncultivable organisms

• not enough visible differences

What characteristics can be used to classify prokaryotes

Physiological, metabolic, morphological

Strain definition and how to distinguish them (3)

Descendants of a single pure culture

• Biovars: strains that are different based on biochemical attributes (metabolic)

• Morphovars: strains that look different (colony/cell morphology under microscope)

• Serovars: strains that have different antigenic properties

Endosymbiotic Theory

• Eukaryotes engulfed bacteria at various points in time and of digesting them, they became stably incorporated into the cell over time

• Analysis of genomes’ mitochondria and chloroplast help prove they are descendants of bacterial cells

Thermophilic ancient lineages

• look like LUCA

• Most deeply-branching lineages

Cyanobacteria

• phyletic group

• contain only bacterial oxygenic photosynthesizers

• can fix nitrogen

• some filamentous, some colonial

• neat differentiation potential

Green Sulfur Bacteria

• Chlorobi

• Anaerobic photolithoautotrophs (get electrons from inorganic substance using light energy and fix carbon)

• Have chlorosomes to contain pigments

• Uses reductive TCA to fix CO2

• When they oxidize sulfur, they accumulate elemental sulfur on the outside of the cell

Spirochaetes

• Bacterium that “drills” in

• Many/most are free-living and anaerobic

• Axial filament: an internal flagella that lets the cell move though highly viscous media

• hides flagella where it can’t provoke antigenic response