Comprehensive Bacterial and Eukaryotic DNA and Transcription Mechanisms

Bacterial Genome

Chromosome + Plasmid

variable and related to lifestyle: free-living large, facultative pathogen intermediate, obligate symbiont pathogen is smallest

Plasmid

Smaller and multiple copies than chromosome

Chromosome

Larger and one copy

Eukaryote vs bacteria chromosome

bacteria more organized in operon, no introns, small number non-coding genes, more compact

Structure of DNA

Double helix, antiparallel 5' --> 3', proofreading and mismatch repair, use opposite strand as template for repair

Nucleotide

Sugar + phosphate + base

Nucleoside

pentose + base

Purines

A-G with double bond

Pyrimidines

C-G with triple bond

RNA OH group

on 2 carbon whereas DNA lack

Chargaff's Rule

%GC content differs among species but is constant in all cells of an organism within a species

Tm

Melting temperature which 1/2 dsDNA have denatured

Thermostability

Can be obtained through more GC, ex: thermophilic organisms higher GC to maintain integrity in hot environment

DNA Replication Components

template strand (semi-conservative), helicase (uses ATP), replication fork, DNA pol lll 5-->3, leading strand 5-->3, lagging strand make Okazaki fragments 3-->5, DNA ligase, Topoisomerase

Techniques Molecular Biology

microarray - protein, northern blot - RNA, southern blot - DNA

Time it takes replicate E coli genome

40 min, and division time 20 min, new DNA replication starts before prior one finishes, multiple replication forks going at once and daughter cells inherit partially replicated chromosomes

DNA pol 1

removes primers so polymerase can continue synthesis, using phosphate from NTP, then ligase reseals after exonuclease activity

OriC

Origin of replication and proceeds in multiple directions, multifork replication allows for 20 min generation time of E. coli

Termination Chromosome Replication

Ter sites act as roadblock for replication

Tus

Termination utilization substance, bind to ter sites and stop replication fork

Topoisomerase

Torsional stress transmit across replication fork, chromosomes between interlinked after replication, and this releases the stress created and decatenation (untangling)

Cis Elements

Elements on same molecule of DNA as gene they replicate

Trans Elements

Elements that can regulate genes distant from the gene they were transcribed on

E. coli partitioning

1 chromosome to each daughter cell

E. coli Segregation

Moving replication chromosome to daughter cells, starts soon after replication

ParA & ParB

Partitioning proteins encoded by Par genes, cis-acting sites, ParB binds pars sites around OriC and ParA binds nonspecific DNA

Central Dogma

DNA --> RNA --> proteins

Difference transcription and translation

Template same but only part of genome transcribed

Translation: RNA poly use ribonucleotides and Uracil, don't need primer, and no proofreading, 5-->3, produce single-strand DNA molecule compared to 1 strand DNA template

Polycistronic mRNA

1 RNA carries genetic info for various genes/ proteins allowing for coordinated expression, related genes in 1 operon

Transcription and Translation in bacteria

Coupled, since no nucleus

Polysome

Multiple ribosome acting on same RNA

Holoenzyme

Core enzyme (5 subunits with catalytic activity but can't bind and start transcription) + Sigma factor (recognize promoter sequence)

Alpha

Enzyme assembly and interaction with regulatory proteins

Beta

Catalytic activity of RNA synthesis

Beta Prime

Catalytic activity and essential DNA binding

Omega

Assembly and stability of RNA polymerase complex

Promoter

Site DNA where transcription initiates, recognized by sigma factors

Transcription start site is not start of coding region of gene, so what is?

5' UTR before coding region

A-subunit

Interact with UP element upstream to improve strength

Sigma 2 Domain

Interact with -10 element to open DNA

Sigma 4 Domain

Interact with-35 element for initial recognition

Promoter Strength

Determine frequency of transcription initiation, relates affinity of RNA polymerase for promoter region

Initiation Transcription

Promoter recognition

Isomerization - rearrangment to allow binding sigma 2 and open -10 element

2 outcome: abort or promoter escape and clearance to elongation

Elongation Transcription

5-->3 synthesis

Termination Transcription

2 types Rho-indep (intrinsic) or Rho dependent

Intrinsic Termination

signal is on same RNA molecule (cis-acting)

-GC rich hairpin loop followed by serine U residues

-hairpin cause RNA pol pause and poly U leads to dissociation with weak DNA-RNA hybrid

Factor-dependent

Requires RNA-DNA helicase, Rho protein (trans-acting)

Rho bind to rut

Rho termination site allow Rho to catch up with RNA polymerase

Rho is ATP-dependent

Rho can't bind rut sites if mRNA being translated so Rho help prevent transcription of mRNA

Sigma Factors E. coli

Diverse set relating to unique environment and stress

Extracytoplasmic Function (ECF) Sigma Factors

Crucial role in signal transduction

Respond to extracytoplasmic stress

Conjunction with membrane-bound anti-sigma factor for quick response

Highly diverse

Transcriptional Regulaton of T6SS by sigma factor

Xanthomonas T6SS transcribed only in presence of sigma factor ECFK, and T6SS prevent ameoba from eating bacteria

Polar effect on gene expression

Some mutation in polycistronic mRNA can effect downstream genes so important to use complement with plasmid to restore gene to prevent downstream effects, often causes premature stop in translation preventing downstream transcription of genes

Advantages to degenerate amino acids

1) Error tolerance: mitigate mutations

2) Translation efficiency tRNAs can recognize multiple codons due to wobble base pairing

3) Evolutionary flexibility: variation without altering protein function

Termination codons:

UAA, UGA, UAG - don't insert an amino acid but prompt the release of polypeptide from the ribosome

Initiation codon

AUG is main one in E. coli but also GUG and UUG

Wobble Base Pairing

Wobble position is anticodon base 34

Codon base 3 is wobble base of codon

Located here bc ribosome monitoring is relaxed at this position, allowing modified bases and nonstandard pairing

Support efficient decoding of the genetic code without compromising fidelity

Reading Frames

6, 3 forward, 3 backward

How ribosome distinguish between start codons

Shine-Dalgarno sequence/ribosome binding start, short region rich in purines that binds to the 3' end of 16S rRNA anti-Shine-Dalgarno sequence

5-10nt upstream of start codon

Polycistronic mRNA each internal ORF has own SD sequence

How is ribosome number determined (70S)

Subunit number is related to configuration and properties of particle (size, shape)

Aminoacyl-tRNA synthetases

Attach an amino acid to a tRNA

Most cells have 20 different ones for each amino acid

Steps of aminoacyl tRNA synthetases

1) amino acid and ATP enter active site of the enzyme

2) AMP is joined to the amino acid, accompanied by release and breakdown of pyrophosphate

3) AMP is displaced by tRNA creating an aminoacyl tRNA

4) aminoacyl tRNA is released from the enzyme

3 Sites of Ribosome

A = acceptor, P = peptidyl, E = exit

Ribozyme

a type of RNA that can act as an enzyme

in ribosome 23S rRNA act as peptidyltransferase enzyme

Translation Steps

formyl group help initiator tRNA enter the P site, which no other aminoacyl tRNA can do

1) aa-tRNA bind to EF-Tu and comes to A site

2) if there is codon-anticodon match, GTP is cleaved and EF-Tu released

3)23S rRNA form peptide bond

4) EF-ts is a guanine nucleotide exchange factor and recycles EF-Tu (GDP >GTP)

5)EF-G catalyzes translocation of the A site tRNA to P site making room on A site, EF-G uses GTP energy to move ribosome forward one codon

6)P site tRNA now moves to E site to exit

Initiation of Translation

Requires 3 initiation factors: IF1, IF2, IF3, and fMet-tRNA

IF3

Helps keep 30S dissociated

IF1

prevents fMet-tRNA from binding to A site

IF2

delivers fMEt-tRNA to P site and promotes the association of 50S subunit

Translation Termination

No tRNA correspond to stop codon so translation stops when they enter A site

termination codons are recognized by release factors

-RF1 & RF2 recognize stop codons and catalyze peptide release

-RF3 is GTPase that removes RF1/2 from ribosome to speed up termination

trans-Translation (tmRNA)

Mechanism to rescue ribosomes that reached the end of mRNA without a stop codon

release factors work only at a termination codon

requires transfer-messenger RNA (tmRNA)

ribosome shifts from translating the mRNA to translating the tmRNA

EF-Tu deliver tmRNA to stalled ribosomes without any codon-anticodon matching

encode a tag sequence of 10 aa that is recognized by ClpXP protease to degrade de polypeptide

tmRNA

both tRNA and mRNA

aminoacylated with alanine

contains ORF that terminates in a stop codon

Difference between DNA damage and mutation

Mutation is what DNA damage becomes when repair fails

Spontaneous DNA Damage

removal of base/depurination

replication error - by polymerase with wrong pairing later fixed with mismatch repair

deamination - amino group removed causing mutation when DNA replicates

Induced

Alkylation - where methyl group inserted

UV light -cause dimers to forms resulting in distortion

Point Mutation

Mutation in a single base pairing during replication

Transitions

Purine for purine or pyrimidine for pyrimidine

Transversions

Purine for pyrimidine

Nonsense Mutation

Results in early stop codon

Missense Mutation

New amino acid

Ames test

Bacterial assay that measures the mutagenic potential of a compound and carcinogenic potential. Works by exposing bacteria that need histidine (an amino acid) to a test substance; if the substance causes a mutation that restores the bacteria's ability to produce its own histidine, the bacteria grow, signaling a positive (mutagenic) result

Mutation generates

Beneficial variants that improve fitness

Neutral variants that drift in populations

Deleterious variants that selection removes

Mutation serve as what

Molecular clocks as they accumulate over time, often use 16S rRNA gene to identify bacterial species as its highly conserved across all bacteria so we can design universal primers, and hypervariable as there are species-specific sequence signatures

Transcriptional Fusion

Promoter of a target gene is placed upstream of a reporter gene, allow to monitor promoter activity by measuring reporter protein without relying on the original gene's translation

Translational Fusion

Combining the coding sequences of two genes (gene of interest, and reporter gene like GFP) in frame to create a single larger fusion protein to track protein location and activity within a cell

Reporter Gene

Gene that makes a phenotype or protein of interest easily detectable to study

Fusion Protein

One protein composed by the fusion of two genes

Mutation

Change in DNA sequence

Mutant

Organism that carries a mutation

Strain

Genetic variant of an organism

Phenotype

Physical or biochemical characteristics of an organism

Genotype

The genetic constitution of an organism

Green Fluorescent Protein (GFP)

Jellyfish obtaining coelenterazine from its diet

Coelenterazine loads into the protein aequorin (calcium activate photoprotein)

Mechanical disturbance causes Ca2+ influx --> Aequorin flashes blue light

GFP absorbs the blue light and re-emits green light

Auxotrophs

Mutation on genes required to make essential metabolite

Luria-Delbruck Experiment (Fluctuation Test)

Random-mutation (Darwinism) vs directed change hypothesis (Lamarckism)

Found direct evidence that mutations occur spontaneously

How to isolate mutant

Screening: process of finding mutants - involves selective conditions to distinguish the mutant vs WT

Positive Selection

Condition which mutant but not WT can replicate - ex: mutant that acquired antibiotic resistance cassette

Negative Selection

Condition which mutant can't grow

Methodologies to create a mutant

Recombineering and allelic replacement

Gene Deletion by Allelic Exchange

Shuttle vector with Ab that replicates in E. coli but not in bacteria you want to delete the gene

Clone mutant gene into a plasmid and introduce vector into bacteria of interest

Need homologous region up and downstream of gene of interest depending on recombination machinery of cell for insertion

Select for Ab contained in vector

Cloning using restriction enzyme

Cut to get sticky ends and ligase back together

Or blunt ends but those are much harder to clone

Difference between DNA replication and PCR

PCR (in vitro) amplifies specific DNA segments using heat cycles, synthetic primers, and thermophilic polymerase, mimicking DNA replication (in vivo) which copies the entire genome within living cells using various enzymes, RNA primers, and consistent body temperature, with DNA replication producing Okazaki fragments while PCR synthesizes only leading strands