MicroBiology Cards Lectures 9-17

Exam 2 Material

Central Dogma

Genes (DNA) —> Transcript (RNA) —> Protein (function

Transcription and Upstream vs Downstream

• First step of gene expression

• reading of DNA template to make —> RNA copy

• guided by un-transcribed DNA regulatory sequences upstream of gene.

Upstream vs Downstream:

Up = left ‘5 , behind gene/going backwards

Down = right ‘3 , before gene

RNA polymerase

Core Enzyme in RNA synthesis

• only one in prokaryotes

Sigma factor

• tells RNA polymerase where to bind in gene

• only needed for initiation of RNA synthesis (not elongation)

• Recognizes promotors by binding to -10 and -35 regions of genes

• guides core enzyme (RNA poly) to initiate transcription (promotor)

Holoenzyme

core enzyme PLUS sigma factor

What happens when there are multiple sigma factors with the cell?

• turns on transcription of different genes responding to different conditions

What sigma factor works all the time? (“housekeeping”)

E.coli sigma factor 70

• recognizes -35 and -10 region

• yellow = the same in all organisms (conserved)

• pink = variable

• TAT box

Can’t be turned off

Initiation of Transcription

1. Sigma factor binds DNA.

2. Sigma factors recruits core enzyme (RNA polymerase) & scans for promotor region

3. Core enzyme unwinds DNA at promoter (Open complex)

4. Sigma factor is released

Then, RNA keeps adding to 5’ - ‘3 strand

Two Termination Mechanisms

• Rho dependent termination (rho enzyme helicase)

• Rho independent termination

Transcription Termination - Rho Dependent

• Rho factor binds to mRNA

• It’s a helicase (unwinds nucleic acids)

• Rho pulls itself to the paused RNA pol

• Breaks polymerase, mRNA off the DNA

Rho Independent Termination

• Polymerase slows at the “pause site”

• GC-rich sequence (tight binding nucleotides) = forms stem loop

• Series of U residues downstream of pause site

• mRNA breaks off the DNA —> polymerase released

Open Reading Frame (ORF)

• start site/codon (AUG) and rest of gene in frame/reading one after another and stop codon

• Each transcript has three possible reading frames (Any 3)

• stop codon in same frame as start codon

tRNA

• grabs/binds amino acids and bring them to ribosome

• have 3 base anticodon —> opposite of what gene says

Ribosomes

• Large rRNA/protein complexes

• 2 subunits 30s and 50s

• 30 S = small subunit

• 50 S = large subunit

• Bind 1 mRNA + 3 tRNAs

Translation in Prokaryotes

Translation and Transcription are coupled here

• Faster than eukaryotes

• Ribosomes bind mRNA while mRNA is being synthesized

• Many ribosomes bind to each mRNA.

• no exons or introns

Shane Dalgarno Sequence

• ribosome binding site on mRNA allowing bind to 30s subunit

Components of Translation Initiation (IF3, IF1,IF2

• IF3: brings “bottom” of ribosome (30s) and mRNA together

• IF1: blocks A site

• IF2: escorts Formylmethionine tRNA (fmet) to —> start codon (AUG)

• tRNA binds directly to P site

Then, “top” (50s) of ribosome docks the “bottom” (30s) subunit

Translation Elongation (3 steps)

1. Aminoacyl-tRNA binds to A site (acceptor site)

2. Peptide Bond formed between new aa and the growing peptide chain in the P site

3. Ribosome shifts down mRNA one codon —> Enzyme exits @ E site

“APE” over and over again

Translation Termination

• stop codon on mRNA enters A site (no tRNA)

• protein releasing factor enters A site —> making everything stop

• Everything clears out of ribosome then ribosome falls apart.

Plasmids - not in humans unique to bacteria

• Extrachromosomal DNA - not part of main chromosome, soak up DNA from environment (usually circular)

• has own origin of replication

• transferred by transformation and conjugation

• randomly segregate to daughter cells during replication

Three transfer processes of Horizontal Gene Transfer

• Transformation: natural transformation: free (naked) DNA taken up form environment —> integrated into genome . Plasmid transformation

• Conjugation: cell to cell contact (bacterial sex)

• Transduction: DNA transfer is mediated by bacteriophage.

Discovery of Transformation

Streptococcus Pneumoniae by Griffth

• Mortality experiment with mice using smooth looking (w capsule virulent) + rough looking (no capsule, nonvirulent) strains

• tested polysaccharide, protein, RNA and DNA —> only DNA from smooth strain conferred virulence to rough strain

Results:

Competent in Gene Transfer: Transformation

Uptake of DNA directly from the environment

• Translocasome in Gram positive bacteria takes up DNA

• Bacteria secrete competent factor —> accumulation of competent factors induces assembly of translocasome located in cell membrane

Competence in Gram Negative Bacteria

• ability of cells to take up foreign DNA

• stress induces competence

• starvation

Gene Transfer by Conjugation

Transfer of DNA from one bacterium to another, following cell to cell contact.

• “bacterial sex”

• initiated by special pilus protruding from donor cell

F factor of Conjugation

• Pilus proteins encode on fertility plasmid or F Factor

• Self transmissible

• Goes from donor —> recipient

• Recipient: transferred DNA recieves a copy of F factor & becomes F+ (Donor)

• “female” becomes “male”

• Recipient is always F-

Rolling Circle Replication

• F plasmid is self replicating —> opens self up and moves along, as it transfers, it copies itself too at same time.

• Has its own “origin of transfer” - single strand nick at oriT

Agrobacterium tumefaciens

• causes crown gall disease

• contains tumor inducing plasmid (Ti) that can be transferred via conjugation to plant cells.

Transduction

bacteriophages (viruses that infect viruses) carry host DNA from one cell to another

• occurs accidentally as an offshoot of the phage life cycle

Restriction Endonuclease

• in recipient cell

• foreign DNA get degraded by this or “chopped” up

What happens to Foreign DNA without DNA methylation?

• Gets destroyed

• Methyl groups work to protect restriction sites

Defense against transferred DNA

• restriction/modification system

• CRISPR - “Clustered Regularly Interspaced Palindromic Repeats” or bacterial immune system against viruses and viral DNA.

• when infected, bacteria cut up viral DNA and insert some pieces into their own genome (to remember virus for future infections)

Spacers

• little virus bits added to bacterial genome —> are transcribed then Cas9 monitors the cell for matching invading DNA.

• bacteria then degrades those matching DNAs to prevent viral infection.

Mutant

organism that is the direct offspring of a “normal” member of species (the wild type/seeing the normal) but is different: containing mutations.

• different than wild type —> “seeing the normal”

• generating new variants in a population

Mutation

change in genetic code, rare.

• not always a good or bad thing

• has to be a heritable change

• changes genotype, may not always change phenotype

Novel functions

provided by mutations

• become “fixed” in populations when they are selected for by natural selection

Spontaneous Mutations

• "An accident, not always perfect”

• arise in low rate of any cells in absence of any added agent

• errors in DNA replication

Induced Mutations

• more common, environment causes to mutate by added mutagens

• physically by sun/heat/radiation

• biological: insertion of transposons

• chemical: compounds that interfere with DNA chemistry

• retroviruses

• smoking/vaping/alcohol

Mutations can be classed based on base pair changes

• changing a letter —> causes a mutation

• slide 29

Silent Mutuation

• can’t see it unless you sequence it

  • mutation does not change the amino acid —> because of degeneracy of genetic code.

Nonsense mutation

mutation produces stop codon

missense mutation

mutation leads to an amino acid change by changing one letter

Frameshift Mutation

changes way we read DNA

• shifting reading frame of gene, changing letter totally (huge changes in genome)

• insertions and deletions

Physical Mutagen - Radiation

• UV and ionizing radiation → kicks off electrons, makes things ions = formation of toxic O2 radicals

• Causing 2 adjacent pyrimidine bases to dimerize —> Thymine Dimers (bridges them together)

• prevents gene transcription and stops DNA replication

Chemical Mutagens and Base Analogs

• Alc and nicotine change code

• Analogs: similar structures to natural bases

• incorporated during DNA replication

  • leads to point mutation bc of incorrect base pairing

DNA modifying agents

changes base’s structure and pairing characteristics

Transposons

• chromosomes sequence is not fixed

• moves DNA segments—> does transposition so moving around

• “jumping genes” Barbara McClintock in Corn

• colors in corn is due to transposons hopping around corn’s genome

• found in all living things

Transposae

“cuts out” insertion/transposable element —> moves it to “better” spot in target DNA

• then ligates the transposable element with the target DNA.

Ames Test

• Bacterial Test to see if chemical can cause mutations or strength of mutagen tested

• take bacteria that is already a mutant, bacteria can’t grow w/o histidine

Procedure:

• take bacteria that can’t grow w/o histidine (can’t grow bc their hisG gene is broken)

• expose bacteria to something you want to see if it is a mutagen

• if there is growth —> there is a mutation

Reversion Mutation (RM) of Ames Test

• Mutagen causes RM of hisG back to functional form —> fixed gene but then causes mutation (carcinogenic)

• more colonies = stronger mutagen

• failing ames test (getting a positive result) = causing mutation in bacteria

• passes = causes cancer

DNA Repair - Proofreading and Photo reactivation

Correction of mismatch by DNA polymerase III during DNA replication

• will cut bases out or fix directly

P. reactivation: deals with damage from sun

• photolyase enzyme binds to pyrimidine dimer and cleaves cyclobutane ring

Excision Repair

Most common damage/mismatch repair

• cut, copy and paste

• cut out bad DNA (wrong base)

• copy from correct strand to replace excised strand

• paste all together by DNA ligase

Nucleotide Excision Repair

• finds mutation that changes shape of DNA

• if there is bend, break or twist —> cuts out (endonuclease) damaged base and reforms it correctly

Base Excision Repair

• changes those mutations that don’t change shape but letter itself.

• ex. A-C binding instead of A-T.

• recognizes and repairs base that don’t distort DNA structure

Methyl Mismatch Repair

• use methyl to determine it’s own DNA

• but base may lose methyl group —> recognizes mismatches missed by proofreading

• Use DNA methylation as an indicator for a newer strand - containing error

SOS Response - “save ourselves”

• fixes mutations all over

• do not want this response on all the time but works fast.

• Extensive DNA damage induces SOS regulatory system

LexA, RecA

• Lex A: keeps SOS response inhibited/off

• RecA: turned on when DNA is damaged, turns off LexA, allows entire response/repair enzymes to turn on

• DNA mutases: error prone DNA polymerases —> repair DNA rapidly but leave many mutations

Double Strand Break repair

• DS breaks are super bad for bacterial DNA.

• causes chromosomal instability —> mutations go everywhere now

Non Homologous End Joining

• when cells see free end of circle —>stick Ku family proteins (caps) sticking to free DNA strand ends

• other proteins including ligases recruited to fix break

• we have this mechanism

lecture 12