Microbial Genetics and Ecology Exam 2 Review

• Micro Exam 2 Review will cover Microbial Genetics and Ecology

Exam 2 Information

• The exam is scheduled for Tuesday, March 11th, during class time
• The exam will be a CANVAS Quiz, using Respondus
• The exam consists of 18 multiple-choice questions (MCQs) and 3 free-response questions (FRQs), with the FRQs worth 27 points per section, totaling 54 points
• The exam duration is 60 minutes

Table of Contents

• Week 5 focuses on Genome structure, central dogma, and expression
• Week 6 covers Regulation and response
• Week 7 concerns Mutation, repair, and variation
• Week 8 explores Microbial Ecology

WEEK 5: DNA and RNA Structure

• DNA is double-stranded and composed of the bases adenine, guanine, cytosine, and thymine, which pair complementarily (A-T with 2 hydrogen bonds, G-C with 3 hydrogen bonds)
• DNA has a sugar-phosphate backbone consisting of phosphodiester bonds
• Supercoiling allows DNA coils to "loosen" to accommodate processes like transcription
• RNA is mostly single-stranded and composed of the bases adenine, guanine, cytosine, and uracil; some RNA can be double-stranded
• Proteins are composed of amino acids linked by peptide bonds and have primary, secondary, tertiary, and quaternary levels of structure

Prokaryotic vs. Eukaryotic DNA

• Prokaryotic DNA is circular, exists in the cytoplasm, lacks telomeres and introns, and consists of only one chromosome with bidirectional replication from a single origin
• Eukaryotic DNA is linear, exists inside the nucleus, contains telomeres and introns, and consists of multiple chromosomes

DNA Replication

• DNA replication is bidirectional in circular bacteria, with the DNA strand unwinding at the replication fork
• DNA replication is semiconservative, each daughter cell has one old strand and one new strand
• Synthesis occurs in the 5' → 3' direction only, resulting in leading and lagging strands (Okazaki fragments)
• DNA polymerase III proofreads the new strand, with exonuclease activity removing mismatched bases from the 3' end
• Helicase unzips DNA by breaking hydrogen bonds between base pairs
• ssDNA binding proteins protect DNA from damage
• Topoisomerases relieve twisting of unwound DNA
• Primase synthesizes RNA primers for DNA polymerase
• DNA polymerase extends the complementary strand of DNA in the 5' → 3' direction
• Ligase forms phosphodiester bonds between Okazaki fragments
• Termination occurs at the termination site on DNA, and topoisomerases can induce a double-strand break (DSB) to separate catenated chromosomes

Transcription

• Transcription produces an RNA strand complementary to a template DNA strand
• Initiation involves RNA polymerase binding to the DNA, with the holoenzyme (core enzyme + sigma factor) and the sigma factor helping the core enzyme find the transcription start site (TSS)
• Elongation: the transcription bubble (RNA:DNA hybrid) moves with RNA polymerase, synthesizing mRNA
• Termination involves RNA polymerase dissociation, which can be intrinsic (stem loop formation) or factor-dependent (Rho protein helicase)
• The result is a messenger RNA (mRNA) strand

Translation

• tRNA has an anticodon complementary to the mRNA codon, and aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to tRNA
• The wobble position is the third position on the codon that allows for flexibility in base pairing
• Ribosomal RNA (rRNA) includes 16S rRNA, which binds to the Shine-Dalgarno sequence and the 3' end of tRNA, and 23S rRNA, which is a ribozyme that catalyzes peptide bond formation
• During Translation:
  • The A (acceptor) site receives tRNA carrying an amino acid
  • The P (peptidyl) site is where tRNA attaches to the growing polypeptide
  • The E (exit) site is where empty tRNA leaves the ribosome
• Initiation begins when the bacterial initiator tRNA (N-formylmethionine-tRNA) adds the start codon (AUG)
• In archaea and eukaryotes, methionine-tRNA adds the start codon
• Elongation: tRNA moves from the A site to the P site to the E site
• Termination occurs at stop codons (UAA, UAG, UGA)
• Bacteria can couple transcription and translation simultaneously for increased efficiency

Post-Translation

• Translocation is the movement of proteins from the cytoplasm to or across the plasma membrane, using the Sec system (general pathway) or the Tat system (folded proteins only)
• Secretion is the movement of proteins from the cytoplasm to the external environment
• Gram-negative bacteria have a 2-step process: translocation to the periplasm by Sec/Tat and secretion across the outer membrane
• A signal peptide is an N-terminal sequence that directs the peptide to a specific route

Gene Structure

• The promoter is the start of the gene where RNA polymerase (RNAP) binds
• The leader is transcribed into mRNA but not translated
• The Shine-Dalgarno sequence initiates translation
• The coding region includes the start codon (AUG) and stop codons (UAG/UAA/UGA)
• Promoter elements include -35 (where sigma recognizes and directs the holoenzyme), -10 (where DNA starts to separate), and +1 (the start of transcription)

Operons

• An operon is a cluster of genes under the control of a single promoter
• The lac operon consists of three genes that metabolize and uptake lactose, and it operates when lactose is present
• The lac repressor binds to the operator, inhibiting transcription when no lactose is present
• The trp operon consists of five genes coding for enzymes to make tryptophan, and it operates in the absence of tryptophan
• When tryptophan is present, it acts as a co-repressor and inhibits transcription

Regulation

• Two-component signal transduction systems involve a sensor kinase phosphorylating itself and then transferring it to a response regulator, which undergoes conformational change and affects transcriptional changes
• Phosphorelays are related to two-component systems
• Sigma factors: RNA polymerase needs sigma to bind to a promoter and start transcription, with alternative sigma factors binding to specific promoters based on cell conditions
• Second messengers are molecules made in response to a first messenger signal, such as cAMP or cyclic dinucleotides
• Catabolite repression represses the use of one catabolite when another is present
• One carbon source is used preferentially (e.g., glucose) until depleted, then after a lag, a second source is used (e.g., lactose), which causes Diauxic growth

Regulation - Additional Mechanisms

• High glucose inactivates adenylate cyclase resulting in no cAMP
• Low glucose triggers cAMP production, it binds to CAP; which leads to RNAP + TS of lac operon, also known as connection to diauxic growth
• Stringent response: triggered by synthesizing pppGpp to counteract environmental stress
• Cyclic dinucleotides: effector molecules for cell cycle progression

Chemotaxis

• Involves movement towards or away from a trigger
• Clockwise rotation leads to tumbling/stopping to change direction
• Counterclockwise rotation allows the cell to run towards a signal

Others

• Quorum sensing: autoinducer signalling molecules increase as population increases
• Restriction-modification: restriction endonuclease cleaves + reduces viral DNA
• Response to viral infection: CRISPR system triggers recognition + cleavage future DNA

Replication in the Three Domains of Life

• Eukarya: Contains linear chromosome, histone, multiple ori's, at least 3 DNAPs, ends with telomeres shortening.
• Bacteria: Contains circular chromosome that uses Bidirectional Replication with one ori.
• Archaea: Contains circular chromosomes that uses 2-4 ori's with no recombinase (similar to bacteria).

Transcriptional Similarities

• Eukarya: Uses Monocistronic mRNA that consists of one mRNA and one promoter.
• Bacteria: Uses polycistronic mRNA with a single RNAP with no introns.
• Archaea: Uses Polycistronic mRNA that has rare introns that share promoter of eukaryotes.

Translational Difference

• Eukarya: 80s ribosome that uses initiation factors to bind to 5' CAP and uses poly-A binding protein for 3’ tail.
• Bacteria: 70s ribosome that has an initiation factor w/tMet tRNA that uses Shine-Dalgarno.
• Archaea: TS/TL is couples, it also uses SD seq!

Regulation of Expression

• Eukarya: The main research is on transcription initiation regulation.
• Bacteria: Have no chromatin or histones, but contain histone-like structures
• Archaea: Has less 2-component regulation but have present histones.

Mutation Types

• Spontaneous mutations: Errors that happen from DNA/genetic mistakes.
• Induced mutations: This happens due to mutagens i.e Thymine dimers d/t to UV radiation
• Forward/Backward: WT → mutant
• Transition mutations: Purine → Purine
• Transversion mutations Purine → Pyrimidine
• Insertion: Adding base/removing base
• Deletions: Lesions is DNA missing a base

Repair Mechanisms

• Proofreading: Corrects bp errors
• Mismatch repair: corrects mismatch repair after replication
• Nucleotide excision repair: System (Uvr) that removes T-T or abnormal alteration
• Base excision repair: fixes base error

SOS Response

• Utilized if it's unable to be repaired by normal means
• Lex A normally bonds to DNA but the prescence of DNA triggers Rec A to destroy Lex A signals repair

Genetic Recombination

• Homologous Recombination is the most common type used with similar nucleotide structure
• Site-Specific Recombination
  • Doesn’t require strong homology
  • Recombination target’s specifics

HGT & Transformation

• Horizontal Gene Transfer: Allows gene movement and adds diversity, through 3 mechanisms.
  • Transformation.
  • Conjugation
  • Transduction

HGT- Conjugation

• How DNA can transfer direct to cell
• The F+ donor cells contains the F factor to express sex pili

Overall Steps

• F+ creates Sex Pili for contact w/ F - cells
• Replication triggers the rolling circle

Hfr Conjugation

• High Frequency Recombination: The F factor allows for the chromosome integration
• Results with a Hfr cell with f - cells (unsuccessful transfer)

Transduction Week 7

• Transfer of genes from viruses that use Bacterial Chromosomes
• Generalize the process

Screening and Cultivation Techniques

• Replica plating allows mutants to cultivate from Wild type d/t the differences
• Auxotrophic and Condition have unique environments
• Study metagenomics, bioinformatics DNA from the environment and map it
• Use of microarray to find levels of mHNA,CHIP to assess the DNA/protein bond

Week 8: Microbial Ecology

• Symbiosis is stable with with both host / symbiont
  • Mutualism: Obligatory with both beneficial relationship.
• Cooperation: Non-obligatory and can loosen relationship
• Antagonism: Negatively affects organism i.e parasitism

Roles of Microbes:

• Allows vascular plant relations
• Provide for Nutrient transport for plant and promotes nitrogen fixation.

Degradation of Organic Matter

• The way we understand that degradation happens
• Oxidation and Nutrient is key
• Allows for a earth support cycle for C, N, P.

Carbon + Nitrogen Cycle

• Carbon Cycle/ N cycle can allow 2 to convert to another
• Nitrogen can allow nitrogen reduction

Nitrogen Cycle

• Multi step progress
• Mineralizing
• Allows the reduction to turn to different process for other processes/genera.

Phosphorus Cycle

• P is limited in soil.
• The cycle can be stimulated with high levels of soil
• P gets absorbed by high amounts of organisms that leads to cycle with the soils and surface.

Fueling Reactions

• Phototroph/ Chemo/ Litho/ Organo/ Hetero/ Autotrophs
• allows to build cells + structures