DNA Replication: Eukaryotic vs. Prokaryotic

Eukaryotic DNA

• Linear chromosomes are located in the nucleus.
• Telomeres and introns are present.
• Eukaryotes have multiple chromosomes and multiple origins of replication.

Prokaryotic DNA

• Circular chromosome is located in the cytoplasm.
• Prokaryotes lack introns and telomeres.
• They possess one chromosome and one origin of replication.

Shared Features: Eukaryotic and Prokaryotic DNA

• Replication fork: DNA unwinds.
• Replicon: A genome segment with an origin, replicated as a unit.
• Semi-conservative replication: Each daughter cell inherits one old and one new strand.

Helicase

• Untwists 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.

Primase

• Synthesizes short RNA primers needed by DNA polymerase.

Clamp Loader Complex

• Helps hold DNA polymerase at the DNA strand.

Tau

• It binds and organizes E. coli replication proteins.

*ORI

• The site where DNA replication starts.
• DnaA proteins attach here.

DNA Replication Steps

• DnaA binds to OriC. Bending and separation of strands occur.
• DnaB, a helicase, separates the strands by disrupting hydrogen bonds between bases.
• Primase then creates RNA primers.
• DNA Polymerase 3 adds bases, synthesizing both leading and lagging strands.
• DNA Polymerase 1 removes primers and replaces the gaps with complementary DNA bases.
• Okazaki fragments are joined by DNA ligase, forming bonds between the strands.
• Exonuclease from DNA Polymerase 3 removes incorrect nucleotides.
• DNA Polymerase 1 removes primers, and DNA Polymerase 3 removes mismatched base pairs from the 3' end.

Leading Strand

• Helicase unwinds DNA, and DNA polymerase adds complementary bases in the 5' to 3' direction.

Lagging Strand

• DNA polymerase can only add to the 3' end.
• RNA primers are added.
• DNA polymerase adds bases to primers in the 5' to 3' direction.
• DNA Polymerase I removes primers.
• Okazaki fragments are joined by DNA ligase.

Central Dogma

• DNA-RNA-mRNA-Protein.
• In prokaryotes, transcription and translation occur simultaneously for efficiency.

Transcription

• mRNA is synthesized using complementary DNA as a template.
• Transcription proceeds via initiation, elongation, and termination.

Bacterial RNA Polymerase

• The core enzyme comprises five polypeptides.

Sigma Factor

• This allows the enzyme to recognize gene starts.

RNA Polymerase Holoenzyme

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

Initiation

• A sigma factor positions the enzyme at the promoter.
• The promoter is a non-transcribed DNA region where RNA polymerase binds to start transcription.
• Sigma factor recognizes and binds 35 bps upstream.
• DNA strands separate at 10 bps upstream.
• RNA polymerase uses the TATA box in eukaryotes and the Pribnow box in prokaryotes.

Elongation

• After binding, RNA polymerase unwinds DNA and proceeds in the 5' to 3' direction.
• The transcription bubble moves with RNA polymerase as it synthesizes mRNA.

Termination

• Occurs when the core RNA polymerase dissociates from the DNA template.
• DNA sequences mark the end of the gene and terminator.
• Termination occurs through intrinsic termination using a stem-loop, or factor-dependent Rho protein (helicase).

Translation

• mRNA is decoded and a protein is produced.
• Amino acids are linked together from the N-terminus to the C-terminus.
• tRNA contains an anticodon complementary to mRNA and attaches to a specific amino acid.
• The third position wobble helps protect against mutations because Codons are degenerate.
• 16s rRNA binds to the Shine-Dalgarno sequence to start 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 and is removed post-translationally.
• Archaea and Eukaryotes: Adds methionine to AUG start codons.
• Eukaryotes do not use the SD sequence to locate the start of translation; instead, they 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 from the A-P-E site on the ribosome.
• Some microbes use selenocysteine and pyrrolysine, encoded by codons that typically function as stop codons but are repurposed in microbes.

Translation Termination

• Termination codons UAA, UAG, and UGA have no corresponding tRNA.

Protein Translocation

• Proteins move from the cytosol to or across the plasma membrane.
• The Sec system is a general pathway that recognizes signal sequences for translocation.
• The Tat system secretes only folded protein and recognizes "twin" arginine residues in the signal sequence to move across the cytoplasmic inner membrane.

Secretion

• Proteins move from the cytoplasm to the external environment.

Signal Peptide

• An N-terminal sequence directs the peptide to a specific route and is removed after maturation.
• Gram-negative processes can either have two steps, translocation to the periplasm via Sec or Tat and secretion across the outer membrane, or one step involving multiple polypeptides that span the periplasm.

Organization of Genes

• Gene: basic unit of genetic information.
• Promoter: Located at the start of the gene.
  • Contains recognition/binding site for RNAP and orients polymerase.
• Coding Region: Starts with codon AUG and ends with stop codon.
• Sigma factor binds 35 bps upstream.
• DNA strands separate at 10 bps upstream.

Operon

• Bacterial & archaeal genes are grouped together and controlled by a single promoter.
• Genes are transcribed together in a single mRNA strand (polycistronic).
• Genes are expressed together or not at all.
• Operons are not common in eukaryotes because they are monocistronic where one gene codes for one RNA.

Operator

• A DNA segment in an operon where the repressor protein binds.
• It controls the expression of adjacent genes and is downstream of the promoter.

Promoter

• Located at the start of a gene to orient polymerase.
• Recognizes and binds RNAP.

RNAP

• RNA polymerase, is an enzyme that catalyzes RNA synthesis.

Binding Site

• A DNA segment in which activator proteins attach upstream of the promoter.

Sigma Factor

• A protein that helps the bacterial RNAP core enzyme recognize the start of the gene to help initiate transcription, while binds to the promoter.

Leading Sequence

• DNA transcribed into mRNA but not amino acids.
• Provides a ribosome binding site for the proper positioning to start reading the codon sequence.

Inducers

• An inducer binds to the repressor protein, preventing its binding to the operator.
• RNAP can then transcribe the genes thus turning gene expression on.

Corepressors

• Corepressors allow repressors to bind to the operator by changing its shape to attach to the operator's DNA sequence, blocking the transcription of a gene off.
• Turns gene expression off.

Alternative Sigma Factor

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

Constitutive Genes

• Housekeeping genes encode enzymes regularly transcribed and translated for a constant supply.
• Other enzymes are only needed under specific conditions, so expression is regulated.

Bacterial Responses to Stimuli

• Transcriptional regulation (mRNA-DNA).
• Translational regulation (regulation on mRNA/ribosome to control protein amount).
• Post-translational modification (modifications on protein to affect activity).

RNA Products Regulated in Each Transcription Step

• Initiation: Activator/repressor binds to the promoter, affecting RNAP binding.
• Elongation: Attenuation.
• Termination: Termination/antitermination loops.

Transcription Factors

• Negative control: A repressor protein blocks RNA polymerase binding.
• Positive control: An activator protein binds to activator sites upstream of the promoter to encourage RNAP binding.

Riboswitches

• A specialized form of transcription attenuation.
• Folding of the RNA leader sequence determines if translation continues or terminates.
• The folding pattern is altered by effector molecule binding RNA.

RNA Thermometers

• Functionally similar to riboswitches, except for translation.
• Small (sRNA), noncoding (ncRNA), antisense RNAs, inhibiting/enhancing termination.
• Chaperone required to the interaction with complementary sequences.

Eukarya

• Dogma moves from nucleus to cytoplasm.
• Monocistronic with typically linear chromosomes and pre-mRNA.
• Post-transcriptional modifications like poly-A tail addition and the 5' cap.
• Splicing occurs.
• There are 3 RNAP types for each type of RNA.
• Complex TFID is required, needing the TATA box.

Archaea

• Combines eukaryotic mechanisms in a bacterial body.
• Eukaryotic aspects: RNAP, TATA box, accessory factors, histones.
• Bacterial aspects: Polycistron, single RNAP, no introns, no splicing.

Operons

• Multiple genes are controlled by one promoter and transcribed together.
• The structure includes a leader first, then a coding region, a spacer (if polycistronic) with more coding regions, and finally a trailer at the 3' end.
• Eukaryotes are monocistronic, with one gene resulting in one RNA and one protein.
• Bacteria and archaea are polycistronic.

Inducible Control

• Turns transcription ON by the presence of specific molecules.
• The LAC operon is induced when lactose produces allolactose via LACL genes, which then binds to the repressor, inhibiting repressor binding and enabling transcription.
• Negative control: genes are expressed unless switched off by a repressor.
• No lactose leads to repressor binding and no transcription.

Repressible Control

• Repressible control turns OFF by the presence of specific molecules.
• The TRP operon is repressed in the presence of tryptophan.
• The operon has five genes coding for tryptophan synthesis and functions in the absence of TRP.
• High TRP concentration leads to repression of transcription.
• Low TRP concentration allows transcription to occur.

Diauxic Growth

• Glucose is used first, and then the switch to lactose occurs when glucose is low.
• This means that the lac operon is active and transcription occurs when lactose is present without glucose or low glucose conditions.

Catabolite Repression

• With existing glucose, bacteria repress the Lac operon, even with lactose.
• Adenylate cyclases convert ATP to cAMP to activate CAP/CRP when glucose is low so that lactose can lactose be used instead.
• On the other Glucose- inactive CAP- cAMP low- LAC is repressed.

Lac Operon Under Lactose Vs Glucose Availability

• Presence of only lactose: allolactose binds to an inactive repressor, allowing transcription with enhanced RNA binding.
• Presence of lactose and glucose: an inactive repressor and inactive CAP result in no transcription.
• Absence of both: an active repressor leads to no transcription.
• Presence of only glucose: glucose is used preferentially, the repressor binds, and CAP remains inactive to cause no transcription.
• Transcription of the Lac operon will only occur when lactose is available, and glucose is not.

Two-Component System

• Signal transduction occurs.
• Sensor kinases will phosphorylate itself, then transfer the phosphate to the response regulator.
• The response regulator undergoes conformational changes.
• For instance, during chemotaxis, the sesnor kinase CheA will transfer phosphate to CheY and CheB.

Alternate Sigma Factors

• RNAP needs a sigma factor to bind a promoter and initiate transcription.
• Alternative sigma factors change many genes' expression, directing RNAP to bacterial promoters.

C-diGMP

• Secondary messengers like cyclic dinucleotides cdiGMP/cdiAMP/cGMP are small molecules produced in response to a signal (first messenger).
• This allows cell cycle progression, biofilm formation, and virulent gene expression.

Stringent Response

• Amino acid starvation results in the cell decreasing tRNA and rRNA production while increasing the transcription of amino acid genes.
• When uncharged tRNA enters ribosomes, the protein RelA makes pppGpp which is the stringent response=stress response.

Chemotaxis

• Movement in response to a chemical stimulus.
• MCP/Methyl-accepting chemotaxis protein and chemoreceptors in the membrane will bind to environmental chemicals, initiating phospho-relay towards CheY, which governs flagella rotation.

Quorum Sensing

• Cell-cell communication.
• AHL binds to the LuxR transcription regulator and activates transcription for all AHL synthase genes, plus proteins for light production.

Mutation

• A heritable change in the DNA sequence can have diverse effects on genes and cellular activity.

Spontaneous Mutations

• Errors in DNA replication results in spontaneous mutations.
• Head-on collisions between the replisomes and RNA polymerase results in spontaneous mutations.
• Spontaneously occurring lesions in DNA results in spontaneous mutations.
• Action of mobile genetic elements results in spontaneous mutations.
• Wild type: The most prevalent form of gene and phenotype.
• Forward mutations: wild types- to mutant form.
• Reversion mutations: Mutant phenotype- to wild type phenotype.
• Suppressor mutation: The wild-type phenotype is restored by a second mutation at a different site than the original mutation.
• Mutations typically occur in regulatory or coding sequences of 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 a pyrimidine with a pyrimidine. This is stable.

Transversion

• Purine substituted for a pyrimidine could cause 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.
• Physical or chemical agents can damage DNA.

Base Analogs

• Structurally similar to normal bases, so mistakes occur when they are incorporated into DNA.

DNA-Modifying Agents

• Alters 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 that will serve as the template strand.

Lesions in DNA

• Lesions in DNA can result in mutations, resulting in a site in DNA where the base is missing.
• This is called an 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 DNA repair mechanisms such as BER.

Genetic Mutation

• A permanent change in the DNA sequence that has already been replicated.

Frameshift Mutation

• This 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

• Mutated non-coding sequences.
• Promoters and enhancers/silencers misregulate genes.
• Mutations result in reduced transcription and a change in gene expression levels.

OFR (Open Reading Frame) Mutations

• Mutations in codon sequences.
• Missense: Alters one nucleotide.
• Nonsense: Changes a codon to a stop codon.
• Frameshift: results in insertion or deletion.
• Tautomerization: Nucleotide substitution causes unique pairing.
• Transition: causes a stable nucleotide substitution.
• Transversion: Purine substitutes a pyrimidine to steric issues.
• Changes in protein can change in function by either gain or loss which can have beneficial to deadly effects.

Bacterial Mutants

• Spontaneous or induced changes in DNA lead to a new phenotype.

Replication Planting Technique:

• Wild type and mutant:
• Growth observed under different conditions.
• Conditional: Different phenotype under certain environmental conditions.
• Autotrophic: Unable to synthesize essential macromolecules like amino acids or nucleotides.

Metagenomics

• DNA is extracted from environmental samples.
• Used for identification of mutants in a microbial community, as well as understanding genetic diseases.

Bioinformatics

• Used for identification and classification.
• Genome Annotation
  • Used to locate genes within a genome and to ID ORFs.
• BLAST (Basic Local Alignment Search Tool)
  • Used to compare gene sequences/assign gene function.

Nucleotide Excision Repair

• This process will remove damage that is distorting the DNA double helix, like thymine dimers.
• It uses the intact strand as a template to synthesize new DNA.
• The aim is to remove lesions and prevent mutations caused by thymine dimers caused by UV light.

Base Excision Repair

• Used to remove minor damage, like oxidized bases.
• Creates AP (apurinic/aplyrimidinic) sites.
• Cleaved by AP endonuclease.
• Uses DNA pol/DNA ligase.
• Works by removing minor damage before replication.

Proofreading

• The correction of errors during DNA replication.
• DNA pol replaces errors with the correct nucelotide.
• Aims at fixing replication errors and reducing mutations.

High Fidelity Repair

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

Error-Prone Repair

• When severe DNA damage occurs, specialized DNA pol replicates over lesions, increasing the mutation rate but potentially allowing for survival.

SOS Response

• Triggered by extensive DNA damage, resulting in a global response.
• RecA proteolyses LexA, increasing repair enzyme production.
• Recombination repair (recA aligns damaged DNA with a 2nd copy of the genome.
• Results in an increased mutation rate but survival despite massive DNA damage.

Recombination Repair

• Used 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

• This moves genes from one organism to another, increasing diversity.
• vs. vertical gene transfer which is when genes are passed down from parent to daughter.

Transformation

• DNA comes from the environment.
• In natural transformation, bacteria lyse, releasing DNA into the environment, 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 to the phage) are mistakenly packaged into a replication phage.

Conjugation

• DNA is transferred via direct contact.
• Rolling circle replication is used so that as the F factor is being transferred, it is also being copied.

Hfr Conjugation

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

Genetic Recombination

• It requires 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.

DNA Microarrays

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

Site-Specific Recombination

• Does not require long sequence homology.
• Recombination occurs at a specific target site.
• Uses the recombinase enzyme.

Transports and Other MGE's

• Transposable genes, also known as "jumping genes," are a mobile Genetic Element (MGE).
• MGE's are genetic elements that move with and between genomes.
  • Simple MGE's use cut and paste.
  • Replicative MGE's use copy and paste.

When Transferring Genetic Info

• HGT and other forms of genetic transfer allow for the uptake of new, modified, or mutated DNA.
• This enables bacteria to acquire specialized genes and potentially develop 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.

Bacterial Ability to Fix Atmospheric Pressure

• Some bacteria can fix atmospheric nitrogen so host plants can use it.
• Some bacteria can form commensal or mutualistic relationships with plants.
• Microbes can act as a plant pathogen and harm the host.

In Deep, Hot Biosphere

• Bacteria and archaea live in biofilms kilometers below the Earth's crust.
• Genomic studies suggest the presence of N2 fixation and denitrification from subterranean.