Microbial Genetics

• This covers key concepts in microbial genetics, including DNA structure, replication, transcription, translation, gene regulation, mutation, DNA repair, horizontal gene transfer, and various genetic analysis techniques.

Eukaryotic DNA

• Linear chromosome located in the nucleus
• Features telomeres and introns
• Contains multiple chromosomes and origins of replication

Prokaryotic DNA

• Circular chromosome found in the cytoplasm
• Lacks introns and telomeres
• Has a single chromosome and one origin of replication

Shared Features of Eukaryotic and Prokaryotic DNA

• Replication fork: Site where DNA unwinds
• Replicon: Genome segment with an origin, replicated as a unit
• Semi-conservative replication: Daughter cells inherit one old and one new DNA strand

Helicase

• Enzyme that unwinds the DNA double helix at the replication fork
• Disrupts hydrogen bonds to facilitate replisome movement

ss DNA Binding Proteins

• Protect DNA from damage

Topoisomerase

• Prevents excessive twisting of DNA during replication

Primase

• Synthesizes short RNA primers needed for DNA polymerase activity

Clamp Loader Complex

• Maintains the position of DNA polymerase on the DNA strand

Tau

• Binds and organizes replication proteins in E. coli.

ORI

• The site where DNA replication begins
• DnaA proteins bind to this site

DNA Replication Steps

• DnaA binds to OriC, causing bending and strand separation

• DnaB (helicase) separates strands by breaking hydrogen bonds

• Primase synthesizes RNA primers

• DNA Polymerase 3 adds complementary bases to synthesize leading and lagging strands

• DNA Polymerase 1 removes primers and fills gaps with complementary DNA

• DNA ligase joins Okazaki fragments, forming bonds between strands

• Exonuclease from DNA Polymerase 3 removes incorrect nucleotides

  • DNA Polymerase 1: Removes primers
  • DNA Polymerase 3: Removes mismatched base pairs from the 3' end

Leading Strand

• DNA polymerase adds complementary bases in a continuous 5' to 3' direction as helicase unwinds the DNA

Lagging Strand

• DNA polymerase adds bases to the 3' end in segments (Okazaki fragments) because that is the only free end available as helicase unwinds
• RNA primers are added, and DNA polymerase adds bases to primers in the 5' to 3' direction
• DNA Polymerase I removes primers, and DNA ligase joins Okazaki fragments

Central Dogma

• DNA -> RNA -> mRNA -> Protein
• In prokaryotes, transcription and translation occur simultaneously, enhancing protein production

Transcription

• Synthesis of complementary mRNA from a DNA template through initiation, elongation, and termination

Bacterial RNA Polymerase

• Core enzyme consists of 5 polypeptides

Sigma Factor

• Enables enzyme to recognize gene start sites

RNA Polymerase Holoenzyme

• Consists of the core enzyme and sigma factor
• Only the holoenzyme can initiate transcription
• Repressors bind to the operator, while activators bind to the enhancer

Initiation (Transcription)

• Sigma factor positions the enzyme at the promoter
• Promoter: Non-transcribed DNA region where RNA polymerase binds to start transcription
  • 35 bps (upstream): Sigma factor recognition and binding
  • 10 bps (upstream): DNA strands separate
• RNA polymerase uses the TATA box in eukaryotes
• The Pribnow box is used in prokaryotes

Elongation (Transcription)

• RNA polymerase unwinds DNA and proceeds in the 5' to 3' direction after binding
• Transcription bubble: Moves with RNA polymerase as it synthesizes mRNA

Termination (Transcription)

• Core RNA polymerase dissociates from the DNA template
• DNA sequences signal the end of the gene and include a terminator
• Mechanisms:
  • Intrinsic termination (stem-loop)
  • Factor-dependent Rho protein (helicase)

Translation

• mRNA is decoded and a protein is produced by linking amino acids from the N-terminus to the C-terminus
• tRNA: Contains an anticodon complementary to mRNA and carries a specific amino acid
• Codons are degenerate at the 3rd position (wobble), which protects against mutations
• Ribosomal RNA contributors include 16s rRNA, which binds to the Shine-Dalgarno sequence (starts protein synthesis) at the 3' end of tRNA
• 23s rRNA is a ribozyme that catalyzes peptide bond formation

Ribosomes

• A (acceptor) site: Accepts tRNA and amino acid
• P (peptidyl) site: Holds tRNA attached to the polypeptide chain
• E (exit) site: Empty tRNA exits ribosomes

Translation Initiation

• rRNA assembles around mRNA, and the first tRNA binds to the start codon
• Bacteria:
  • Adds to the AUG start codon downstream of the Shine-Dalgarno sequence
  • Removed post-translationally
• Archaea and Eukaryotes:
  • Adds methionine to AUG start codons
  • Eukaryotes do not use the SD sequence but instead use the 5' cap

Translation Elongation

• Ribosomes move along mRNA and read each codon, tRNA brings corresponding amino acids to elongate the polypeptide chain
• tRNA moves A-P-E site on ribosome
• Some microbes use two rare amino acids: selenocysteine and pyrrolysine, both encoded by codons that typically function as stop codons

Translation Termination

• Termination codons: UAA, UAG, and UGA do not have corresponding tRNAs

Protein Translocation

• Movement of proteins from the cytosol to or across the plasma membrane
• Sec system: General pathway recognizing signal sequences within a protein to translocate it
• Tat system: Secretes only folded proteins and recognizes "twin" arginine residues in the protein signal sequence

Secretion

• Movement of proteins from the cytoplasm to the external environment

Signal Peptide

• N-terminal sequence directing a peptide to a specific route and gets removed after maturation
• Gram-negative process:
  • Can have two steps (translocation to periplasm via Sec or Tat, then secretion across the outer membrane)
  • Can be one step (involves multiple polypeptides that completely span the periplasm)

Gene Organization

• Gene: Basic unit of genetic information
• Promoter: Located at the start of a gene
  • Contains recognition and binding site for RNA polymerase, which orients the polymerase
• Coding Region: Produces codon AUG and ends with a stop codon
• Sigma factor binds 35 bps upstream
• DNA strands separate at 10 bps upstream

Operon

• Typically found in bacterial and archaeal genes
• Operons are groups of genes that work together controlled by a single promoter
• Transcribed together on a single mRNA strand (polycistronic)
• Expressed together or not at all
• Uncommon in eukaryotes (monocistronic- one gene RNA)

Operator

• DNA segment in an operon where the repressor protein binds
• Controls the expression of adjacent genes
• Located downstream of the promoter

Promoter

• Located at the start of a gene
• Orients polymerase to recognize and bind RNA polymerase

RNAP

• RNA polymerase
• Enzyme that catalyzes RNA synthesis

Binding Site

• DNA segment where activator proteins attach upstream of the promoter

Sigma Factor

• Protein that helps bacterial RNA polymerase core enzyme recognize the promoter at the start of a gene
• Binds to the promoter and helps initiate transcription

Leading Sequence

• A sequence in DNA transcribed into mRNA but not amino acids
• Provides a ribosomal binding site and facilitates proper positioning to start reading the codon sequence of the mRNA

Inducers

• Bind to the repressor protein, preventing it from binding to the operator
• RNA polymerase can then transcribe the genes, turning gene expression on

Corepressors

• Allows repressors to bind to the operator by binding to the repressor protein, changing its shape
• Activates a repressor, allowing it to bind to the operator, turning gene expression off

Alternative Sigma Factor

• Allows bacteria to change the promoter specifically to the core RNA polymerase
• Allows bacteria to express genes that help them adapt to environmental and metabolic stimuli

Constitutive Genes

• Housekeeping genes
• Encode enzymes that are regularly transcribed and translated for a constant supply
• Other enzymes are only needed under specific conditions (expression is regulated)

Bacterial Regulation of Response to Stimuli

• Transcriptional regulation (mRNA-DNA)
• Translational regulation (regulation on mRNA/ribosome to control the amount of protein being made)
• Post-translational modification (modifications on proteins to increase/decrease their activity, e.g., cleavage or phosphorylation)

RNA Product Regulation During Transcription

• Initiation: Activators/repressors bind to the promoter and affect the binding of RNA polymerase
• Elongation: Attenuation
• Termination: Termination/antitermination loops

Transcription Factors

• Negative Control:
  • Repressor protein
  • Binding to the operator blocks RNA polymerase from binding
• Positive Control:
  • Activator protein
  • Binding to activator sites upstream of the promoter encourages RNA polymerase to bind

Transcription: Riboswitches

• Specialized form of transcription attenuation
• Folding of the RNA leader sequence (riboswitch) determines if translation will continue or terminate
• The folding pattern alters in response to effector molecule binding to RNA

Translation

• RNA Thermometers:
  • Function similarly to riboswitches, except for translation
• Small (sRNA), non-coding (ncRNA), antisense RNAs:
  • May inhibit or enhance termination
  • Require a chaperone to promote interaction with complementary sequences

Eukarya

• Central dogma moves from the nucleus to the cytoplasm
• Monocistronic genes, typically linear chromosomes, pre-mRNA
• Post-transcriptional modifications such as poly-A tail addition and 5' capping
• Splicing
• Three RNA polymerase types for each RNA type
• Complex TFID requiring the TATA box

Archaea

• Eukaryotic mechanisms in a bacterial body
• Eukaryotic Aspects:
  • RNA polymerase
  • TATA box
  • Accessory factors
  • Histones
• Bacterial Aspects:
  • Polycistronic genes
  • Single RNA polymerase
  • No introns or splicing

Operons

• Multiple genes are controlled by one promoter and transcribed together
• Organized into 3-4 regions: a leader first, then the coding region, then a spacer (if polycistronic) with more coding regions, and finally a trailer at the 3' end
• Eukaryotes are monocistronic, where 1 gene results in 1 RNA, resulting in 1 protein
• Bacteria and archaea are polycistronic

Inducible Control

• Turns transcription ON in the presence of specific molecules
• The LAC operon is induced in the presence of lactose
• Presence of lactose induces the transcription of genes that utilize lactose
• Allolactose will be made via LACL genes and bind to the repressor
• No repressor binding = transcription
• Negative control: Genes are expressed unless switched off by a repressor
• No lactose = repressor binds = no transcription

Repressible Control

• Turns OFF transcription in the presence of specific molecules
• The TRP operon is repressed in the presence of tryptophan
• The operon itself has 5 genes, coding for the synthesis of tryptophan, which function in the absence of TRP
• When TRP is present, it will act as a co-repressor
• No need to make more TRP if you already have it
• Low TRP -> transcription will occur

Diauxic Growth

• Glucose is preferentially used first, before switching to lactose when glucose is low
• The lac operon is active, and transcription occurs when there is lactose and no/low glucose

Catabolite Repression

• When glucose is present, bacteria repress the Lac operon even if lactose is available, so that glucose can be used
• Adenylate cyclases convert ATP to cAMP in order to activate CAP/CRP
• Occurs when glucose is low, so lactose can be used instead
• When there is ample Glucose, CAP is inactive, cAMP is low, and LAC is repressed

Lac Operon Under Lactose vs Glucose Availability

• Lactose: Allolactose binds to inactive repressor -> transcription (YES) -> enhanced RNA binding (YES)
• Lactose and Glucose: Inactive repressor, inactive CAP -> transcription (NO)
• Neither: Repressor is active -> transcription (NO)
• Glucose: Used preferentially; no lactose, so the repressor binds, CAP is inactive -> transcription (NO)
• Overall, transcription of the Lac operon occurs under lactose availability and glucose non-availability

Two-Component System

• Signal Transduction
• Sensor kinases phosphorylate themselves and then transfer the phosphate to the response regulator
• The response regulator undergoes conformational changes
• Example: Chemotaxis: The sensor kinase CheA transfers phosphate to CheY and CheB (response regulators), eliciting changes

Alternate Sigma Factors

• RNA polymerase needs a sigma factor to bind a promoter and initiate transcription
• Alternative sigma factors change the expression of many genes, directing RNA polymerase to specific subsets of bacterial promoters

C-diGMP

• Secondary messengers
• Small molecules produced in response to a signal (e.g., the first messenger)
• Example: Cyclic dinucleotides (cdiGMP/cdiAMP/cGMP)
• Allows for cell cycle progression, biofilm formation, virulent gene expression, etc.

Stringent Response

• Amino acid (aa) starvation results in the cell decreasing tRNA and rRNA production while increasing the transcription of aa genes
• When uncharged tRNA enters ribosomes, protein RelA makes pppGpp

Chemotaxis

• Movement in response to a chemical stimulus
• MCP/Methyl-accepting chemotaxis protein + chemoreceptors in the membrane
• Binds to environmental chemicals, initiating a phospho-relay toward CheY, which governs flagella rotation

Quorum Sensing

• Cell-cell communication
• AHL (Acyl-homoserine lactones) binds to the LuxR transcription regulator and activates transcription for all AHL synthase genes, plus proteins for light production

Mutation

• Heritable change in the DNA sequence that can have various effects on genes and cellular activity

Spontaneous Mutations

• Result from:

  • Errors in DNA replication
  • Head-on collisions between replisomes and RNA polymerase
  • Spontaneously occurring lesions in DNA
  • The action of mobile genetic elements

• Wild Type: Most prevalent form of a gene and phenotype

• Forward Mutations: Wild type -> mutant form

• Reversion Mutation: Mutant phenotype -> wild-type phenotype

• Suppressor Mutation: Wild-type phenotype restored by a second mutation at a different site than the original mutation

• Mutations normally occur in regulatory or coding sequences, tRNA, and rRNA genes

Tautomerization

• Nitrogenous base of nucleotides shifts to a tautomeric form, allowing for unique base pairing to occur

Transition

• Replacement of a purine with a purine or pyrimidine with a pyrimidine (stable)

Transversion

• A purine is substituted for a pyrimidine (steric problems)

Insertion and Deletion

• Occur at short stretches of repeated nucleotides
• Pairing of the template and new strand can be displaced

Induced Mutations

• Result from exposure to a mutagen
• Mutagens: Physical or chemical agents that damage DNA

Base Analogs

• Structurally similar to normal bases
• Mistakes occur when they are incorporated into DNA

DNA-Modifying Agents

• Alter a base, causing it to pair incorrectly

Intercalating Agents

• Distort DNA to induce single nucleotide pair insertion/deletion

Base Pairing Permanent Issue

• When DNA replication is performed on a strand containing a mutation before it can be repaired, the mutation becomes part of a new strand, which will serve as the template strand

Base Pairing Permanent Issue

• Lesions in DNA can result in mutations as they lead to a site in DNA where the base is missing (AP site = apurinic site or apyrimidinic site, depending on the nature of the missing base)

Point Mutation

• A single base pair in a genome is altered, added, or deleted
• Can still be repaired by the DNA repair mechanism (BER)

Genetic Mutation

• Permanent change in the DNA sequence
• Has already been replicated

Frameshift Mutation

• Shifts the reading frame of a DNA sequence by adding/deleting nucleotides
• The number of nucleotides should not be divisible by 3 (because that does not shift the reading frame)

Regulatory Region Mutations

• Non-coding sequences
  • Promoters
  • Enhancers/silencers: Misregulation of genes
• Reduced transcription
• Change in levels of gene expression

OFR Mutations

• Codon sequences
  • Missense: Alters 1 nucleotide
  • Nonsense: Codon to stop codon
  • Frameshift: Insertion/deletion
  • Tautomerization: Nucleotide substitution -> unique pairing
  • Transition: Stable nucleotide substitution
  • Transversion: Purine substituted for a pyrimidine (steric issues)
• Changes in protein: Change in function (lose/gain)
• Wide range of effects (beneficial - deadly)

Bacterial Mutants

• Spontaneous or induced changes in DNA and new phenotypes

Replication Planting Technique

• Wild-type vs. mutant
• Growth under different conditions
  • Conditional: Different phenotypes under certain environmental conditions
  • Auxotrophic: Unable to synthesize essential macromolecules (aa, nucleotides)

Metagenomics

• Extract DNA from environmental samples
• Identification of mutants in the microbial community
• Genetic diseases

Bioinformatics

• Identification/Classification
• Genome Annotation:
  • Locate genes within the genome
  • Identify ORFs (Open Reading Frames)
• BLAST (Basic Local Alignment Search Tool):
  • Compare gene sequences
  • Assign gene function

Nucleotide Excision Repair

• Removes damage (thymine dimers) causing distortion of the DNA double helix
• Uses the intact strand as a template to synthesize new DNA
• Removes lesions and prevents mutations caused by thymine dimers (caused by UV light)

Base Excision Repair

• Removes minor damage (oxidized bases)
• Creates AP (apurinic/aplyrimidinic) sites
• Cleaved by AP endonuclease
• DNA polymerase/DNA ligase
• Removes minor damage before replication

Proofreading

• Correction of errors during DNA replication
• DNA polymerase replaces incorrect nucleotides with the correct ones
• Fixes replication errors and reduces mutations

High-Fidelity Repair

• 3'-5' exonuclease activity
• Corrects mismatches during DNA replication
• Accurately reduces replication errors

Error-Prone Repair

• Occurs when severe DNA damage occurs
• Specialized DNA polymerases replicate over lesions
• Increased mutation rate but may allow for survival

SOS Response

• Extensive DNA damage: Global response
• RecA proteolyzes LexA, increasing repair enzyme production
• Recombination repair (RecA aligns damaged DNA with a 2nd copy of the genome)
• Increased mutation rate but survival of massive DNA damage

Recombination Repair

• Occurs when DNA has both bases damaged
• RecA recognizes and aligns damaged DNA with an undamaged copy
• Corrects double-stranded breaks (mismatched/excision not able to repair)

Horizontal Gene Transfer

• Moving genes from one organism to another to increase diversity
• Vertical gene transfer: Genes are passed down from parent to daughter

Transformation

• DNA comes from the environment
• Natural transformation: Bacteria lyses, releasing DNA into the environment, and DNA that comes into contact with the competent cell is introduced
• Plasmids can also be transferred between bacteria

Transduction

• Transferring bacterial genes using viruses
• A phage infects bacteria, and during viral assembly, host fragments (similar in length as the phage) are mistakenly packaged into the replication phage

Specialized Transduction

• Lambda phage integrates at a specialized attachment site (att) in bacterial genomes

Conjugation

• Rolling circle replication: As the F factor is being transferred, it is also being copied

Hfr Conjugation

• Hfr = bacterium with the F factor plasmid incorporated into its chromosome
• The F factor tries to transfer itself, attempting to drag the rest of the genome along with it (unsuccessfully)
• The F factor can also leave the bacterial chromosome and continue its status as an autonomous plasmid

Genetic Recombination

• Long regions of DNA with similar nucleotide sequences
• A double-stranded break occurs, and crossing over leads to the final product
• RecA carries out the process

Site-Specific Recombination

• Does not require long sequence homology
• Recombination at specific target sites
• Uses the recombinase enzyme

Transports and Other MGE's

• Transposable genes "jumping genes" are a mobile Genetic Element (MGE)
• These are genetic elements that move within and between genomes
  • Simple: Cut and paste
  • Replicative: Copy and paste

Transferring Genetic Info

• HGT and other forms of genetic transfer allow for the uptake of new/modified/mutated DNA
• This enables the bacteria to acquire specialized genes, and potentially resistance to antibiotics
• Immunity genes protect microbes from antibiotics
• Natural selection also plays a role
• Resistance genes are found on: R plasmids, pathogenicity islands, chromosomes, and transposons

DNA Microarrays

• Determines what genes are expressed at a given time
• Arrays: Solid support grid that DNA is attached to and organized on
• Each spot/probe = a single gene
• Probe: PCR products from the gene of interest

RNA Sequencing

• Quantifies mRNA by measuring the reads that match each gene
• Reverse transcription converts mRNA to cDNA
• cDNA is then sequenced using next-gen sequencing