Microbial Genetics Exam II

TFIIB

recruits pol II to TBP, sets transcription start site

5' cap

- 7-methylguanylate triphosphate cap added 5' to 5'
- protects the nascent mRNA from degradation and assists in ribosome binding during translation
-needed for translation initiation
-added by guanylyl transferase anf then methylated by a methyl transferase

synaptonemal complex (SC)

Structure made of multiple proteins that pairs homologs together

Yeast tetrads

when a yeast diploid goes through meiosis, the four haploids are stuck together

complementation test

a test for determining whether two mutations are in different genes (they complement and are in the same complementation group) or the same gene (they do not complement)

Mutant hunt steps

1. test to see if the mutants are dominant or recessive by crossing them with the WT

2. test if the mutant phenotype is due to a mutation in a single gene by doing tetrad analysis and looking for 2:2 ratio

3. Test among the 100 mutants to see how many genes are causing the phenotype by doing a complementation test and assigning complementation groups

4. Now you can move on to identifying your gene of interest

DNA pol I

removes the RNA primer and replaces it with DNA, important in repair/clean up

oriT

origin of transfer

broad host range plasmids

The fact that some plasmids can reproduce in many different and not related species of bacteria, larger and typically bring their own replication machinery

ex. RSF1010

resT protein

the protein that resolves dimers particularly in linear plasmids

Col E1 replication system

copy number is determined by RNA I and RNA II. Depending on the ratio of the two replication can be activated or inhibited.

RepA protein

helps with replication of R1 plasmid, determines the copy number(more=more replication)

self-transmissible plasmid

encodes all the functions needed for its own intercellular transmission by conjugation

TraI

-relaxase; part of the relaxosome complex responsible for strand cleavage and recognition

-cleaves a oriT and become covalently linked to the DNA via phosphotyrosine linkage

-while bound to DNA, it transfers the DNA through the MPF

regulation of f transfer

F uses proteins such as FinO to regulate how much it transfers to different bacteria. the first F plasmid discovered had a mutation that made it transfer a lot more than it should

-all the genes needed for it are organized in a operon after the oriT

-when F first enters the cell there isn't any FinO so TraJ is expressed but once the plasmid is established FinO and P shut transfer down

Firmicute(gram positive) DNA uptake

A channel in membrane forms and dsDNA is cleaved by an endonuclease and the ssDNA pieces can pass through the channel into the cell

ComEA

a protein that binds dsDNA in G+ bacterial DNA uptake

ComFA

ATP dependent translocase that transfers DNA into the cell

ComA

a channel that allows DNA enter the inner membrane

specificity of DNA uptake

-some bacteria(Neisseria and Haemophilus) preferentially take up DNA from related bacteria

-helps to avoid take up of foreign DNA or phage DNA

Congression

uptake of 2 different DNA fragments into a cell

ComA(B. subtilis)

a response regulator, gets signal from ComP

ComQ

Digests ComX

Artificially induced competence

chemically induced, electroporation, protoplast formation

Class I transposons

-includes insertion sequences(IS elements)

-~1kb long

-contain gene tnpA

-some use cut+paste method others use replicative method, some use both

-usually don't carry antibiotic reistance

-can have several copies/cell

2 step movement of class II transposons

Step 1: replicative transposion

Step 2: site specific recombination by TnpR resolves the cointegrate into the donor and the recipient, each with one copy of the transposon

RNA polymerase II

-transcribes mRNA

-has 12 subunits(Rbp1 is the largest)

-needs transcription factors to recognize the promoter

RNA polymerase II CTD

-the C terminal domain on Rpb1

-starts out un phosporylated

-tandem repeats of 7 amino acids(YSPTSPS)

-humans have 53 repeats, yeast have 26 and flies have 43

-phosphorylation and dephosphorylation of these amino acids changes as transcription goes along which changes the proteins that can bind

eukaryotic core promoter

-TATA box(only actually in 10% of promoters)

-Initiator(INR)

-downstream promoter elements(DPEs)

TATA box

-A DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex.

-only really found in ~10% of promoters

-TATAWR, W=A/T and R=A/G

Enhancers

-A DNA sequence that recognizes certain transcription factors that can stimulate transcription of nearby genes.

-Can be very far away from the gene it enhances

-yeast don't have them

upstream activation sequence (UAS)

sequences of DNA upstream of the promotor that when enhancer proteins are bound, they communicate with the promotor with signals to initiate transcription

Coregulators

regulatory proteins that connect RNA pol to enhancers and other elements needed for transcription to occur

general transcription factors

Proteins that assemble on the promoters of many eukaryotic genes near the start site of transcription and load the RNA polymerase in the correct position.

TFIID

the first general transcription factor to bind the promoter, binds to the TATA box through the TATA binding protein (TBP). Also has TBP associated factor(TAFs)

TFIIF

binds pol II and involved in pol II recruitment

TFIIE

binds the promoter and helps open DNA

TFIIH

-DNA helicase, opens up DNA to start transcription

Kin28

the kinase attached TFIIH that phosphorylates the CTD of Pol II

Ctk1

another kinase that phosphorylates the second amino acid in the repeat chain

poly A tail

-added co-transcriptionally

-around 80-250 A residues

-to be added the ser2 must be phosphorylated

-important for translation

-protects RNA from degradation

cleavage-polyadenylation stimulating factors(CPSF)

bind to the pol II CTD and cleaves the 3' end of pre-mRNA before PAP comes in and adds adenosine residues

PolyA polymerase (PAP)

adds A residues to the 3' end of the cleaved transcript

PolyA binding Protein (PABP)

binds the polyA tail and protects it

XRN2

5' Exonuclease; this process involves the exonuclease catching up to the pol II and terminating the transcription.

replica plating

A method of inoculating a number of solid minimal culture media from an original plate to produce the same pattern of colonies on each plate

parental ditype (PD)

a tetrad in which all four spores are parental types (two spores of each of the two parental types possible)

non-parental ditype (NPD)

a tetrad where all 4 spores only have recombinant types, produced by 4-strand double crossover

tetratype (TT) tetrad

a tetrad with 4 different types, 2 are parental and 2 are recombinant

centromere mapping

considers a gene locus and asks how far it is from its centromere, can only be done for unlinked genes

Fluctuation test

a test used in microbes to establish the random nature of mutation or to measure mutation rates

Things to do before a mutant hunt

-you want to have haploids

-do a fluctuation test to make sure you're not working with many copies of the same mutant

-choose both mating types

Cloning by complementation

1. Transform Gal- mutant with a plasmid library that contains an auxotrophic mutant and the WT of your gene of interest

2. replica plate onto media with the experimental condition(has auxotrophic marker)

3. Select for the ones that lived on the Gal media since those were rescued by the plasmid.

4. Transform E. coli with the plasmid to grow more copies

5. Then retransform into the original Gal1 mutant to see if it was really the plasmid causing the Gal+ mutant

6. Now you can sequence the plasmid and use the info you have to identify candidate genes

7. you can then take these candidates, place them on a plasmid and grow them. Next you'll place them on Ura- media and then screen them on Gal media

8. the colonies that live on the Gal media(ie they complement)

Bulk segregant analysis

1. cross your mutant with a WT strain

2. Do tetrad analysis. Now you can sort them into groups with and without your mutation.

3. you can pool them together and then send for WGS. It'll be easy to tell which DNA is for your mutant and which is for the others because of the large numbers of each

DNA pol II

repair of DNA damage

DNA pol III

major replication enzyme

Key features of plasmids

-naturally occurring

-vary in size (1-10kbp)

-Mostly circular

-variable copy number

-often carry genes for antibiotic resistance

-have their own ori, partitioning and dimer resolution machinery

-Naming convention: p for plasmid and then some letters and numbers

circular plasmids vs supercoiled plasmids

circular plasmids migrate slower in a gel compared to supercoiled ones

oriV

origin of replication in a plasmid, site where replication of a plasmid begins

narrow host range plasmids

The fact that only a limited species of strains of bacteria are conducive to a certain plasmid's replication ability, typically are smaller bc they need to use host machinery

ex. ColE1

theta replication

-plasmid replication method
-can be either unidirectional or bidirectional

2 step rolling circle replication

Step 1: Rep protein binds at the double strand origin and nicks the DNA and forms a phosphotyrosine linkage to the DNA. The 3' end serves as a primer so DNA Pol III can synthesize new DNA. Once replication ends the DNA is sealed by breaking the phosphotyrosine linkage with Rep. This produces one dsDNA and one ssDNA molecule

Step 2: Part of the single stranded origin folds to make dsDNA which RNA pol recognizes and makes a primer. Now DNA pol III comes in and finishes synthesizing the plasmid

double stranded origin(DSO)

part of the oriV of plasmids, where rep makes a single strand nick and binds

single stranded origin(SSO)

part of the oriV, forms a dimer that RNA Pol recognizes to make a primer

linear plasmid replication

-Linear plasmids typically have closed ends

-replication starts at the oriV and proceeds bidirectionally

-produces a dimeric plasmid with resT sites which cleaves at inverted repeats and resolves the dimer to give 2 strands of dsDNA

plasmid copy number

-the average number of plasmid molecules in a single cell

-most naturally occurring plasmids have copy numbers of 1-50/cell. Mutants can be made to have 100+ copies/cell

low copy number plasmids

stringent replication systems(tightly regulated)

high copy number plasmids

relaxed replication systems

plasmid incompatibility

-plasmids with similar replication or partitioning control are incompatible because they compete for the same replication machinery.

-the total copy number is maintained but the ratio of the two is not

-are in the same inc (incompatibility) group

incompatibility(inc) groups

a group of plasmids that cannot coexist stably in the same organism

ColE1 plasmid

-a plasmid present in some E. coli strains, 6.6kbp

-origin is present in many plasmids we work with in the lab

-doesn't use a Rep protein to replicate

ColE1: RNA I

an inhibitor of replication, forms a kissing complex with RNA II which inhibits its function as a primer

ColE1: RNA II

a short RNA that serves as a primer for DNA replication, forms an RNA-DNA hybrid at oriT which is processed by RNase H

Rop protein

mediates the kissing complex formed by RNA I and II

CopA

an antisense RNA that inhibits RepA expression, when copy number gets to high it inhibits RepA by pairing with the repA mRNA

CopB

represses the repA promoter. Not present when the plasmid first enters the cell so copy number is high at the beginning

plasmid dimer resolution: XerCD

a site specific recombinase used by ColE1 which recognizes cer(like dif) sites

LoxP-Cre recombination system

similar to the XerCD/cer system. Site specific recombinase Cre recognizes loxp sites

Plasmid partitioning: R1

uses parC/parM/parR system

parC

a site on the plasmids that parR binds to, centromere like

parR

binds to parC site on the plasmid. parM forms a filament that pushes this protein that is bound to the two plasmids apart

parM

bound to ATP it forms a filament that pushes plasmids apart. conversion of parM-ATP to parM-ADP makes the filaments depolarize leaving the plasmids at opposite poles of the cell

plamsid toxin antitoxin systems

-plasmids often encode a toxin and and antitoxin, this helps maintain the plasmid in the population

-when the cell divides if it doesn't get the plasmid the antitoxin it received will degrade(bc its unstable) and the toxin will kill the cell so any cell without the plasmid dies

-helps with phage defense because phages inactivate the antitoxin so the cell dies and the phage doesn't get to replicate

Key features of plasmid cloning vectors

-need an origin of rep

-selectable markers(ie antibiotic resistance)

-useful and unique restriction sites

Conjugation in bacteria

Bacterial cells can join together and pass plasmid DNA from one bacterial cell to another. This process can take place between bacteria of different species and is of concern in terms of passing plasmid-located genes for antibiotic resistance.

mobilizable plasmids

-Contain origin of replication but no transfer genes
-Can only be transferred at same time as conjugative plasmid

f plasmid

-low copy number(1-2/cell)
-100kbp
-self-transmissible plasmid
-50% of genes are tra genes
-has several transposon inserts

F plasmid transfer

-F+ cell produces F pilus that connects it to F− cell

-Transfer of F plasmid occurs through conjugation bridge

-F plasmid copied through rolling circle replication

-The end result is two F+ cells

TraA

pilin protein in F transfer

TraD

coupling protein

TraB and TraK

form a pore that allows MPF to extend through the membrane

relaxasome

-facilitates bridging of cells and transfer of DNA

-made up of relaxase TraU, TraM and TraY. IHF from the bacteria is also needed

-in some plasmids(not F) a DNA primase is also transferred to promote lagging strand DNA synthesis

Mating pair formation (Mpf)

The structures that bring the donor in contact with the recipient and the bridge the DNA goes across (eg sex pilus and bridge)

DNA transfer and conjugal Replication(Dtr)

part of the F transfer apparatus

TraJ

activates the promoter for traY and traX

TraX and Y

genes needed for transfer of F

FinO

-binds to FinP and stops traJ expression which shuts down traX and traY expression and so transfer stops

-in the first discovered F plasmid there's an transposon in the gene so transfer is constitutively on

FinP

sRNA inhibits traJ mRNA translation and stimulates degradation by binding with FinO

mobilizable plasmids transfer

-need a self transmissible plasmid to transfer

-many mobilizable plasmids carry their own Dtr systems(mob genes)

-couple protein is critical for communication btw MPF and Dtr systems

-if relaxase of the mob plasmid is recognized by the transmissible plasmid then if can be tranferred

Hfr strain

Where an F plasmid is integrated into the bacterial chromosome at a specific site