MCB 2050 midterm content

to express the proper gene in the proper cell at the proper time- as cells differentiate different gene expression is required

Purpose of gene control in eukaryotes

RBC and gametes

what cells have a different genome than the rest of the organism

epigenetic

what kind of regulation is chromatin mediated

common genes that are always on ex. actin, tubulin

what genes are always open on chromatin

heterochromatin (not accessible for transcription- inactive less used genes, darker more compact regions)

what type of chromatin has transposons, telomeres, centromeres?

euchromatin

less dense chromatin where frequently transcribed genes are found

1- pre-rRNA

2-mRNA, (snRNA, siRNA, miRNA)

3-tRNA, (some rRNA, signal recognition particle)

RNA pol 1

RNA pol2

RNA pol3

crystallography

yeast pol 2 resolved by _______ has homology to all eukaryotes

12 (12 genes) RPB 1-12

how many peptides in RNA pol 2

RBP1

clamp domain RPB____ is positioned over DNA and closes by bridge

Mg2+

RNA synthesis takes place in catalytic centre with what metal ion?

5’ methyl guanosine

synthesized RNA is immediately capped by _____

B and B’

RPB1 and RPB2 are similar to what prokaryotic RNA pol domains

24, 52

CTD in yeast has _____ repeats and in mammals has ____ repeats

YSPTSPS

CTD sequence

TF2B recognition

function of BRE

-31 to -26

TATA position

-2 to +4

Inr position

TATA

is Inr or TATA more conserved

+28 to +32

position of DPE

at initiation site usually on A, on coding strand

where does transcription start?

helicases, kinases (phosphorylate), EF, protiens taht move nucleosomes out of the way

other factors of initiation

1- TF1A, TF1B

2- TF2A, TF2B, TF2D, TF2E, TF2H and others

3- TF3B, TF3S

GTFs

polymerase chain reaction, amplifies DNA by cycles of denature and renature

PCR definition and purpose

Taq polymerase, thermostable

what polymerase used in PCR and why

1- two primers anneal to DNA ends (50-60)

2- Taq pol synthesises new strands from 3’ end (72)

3- newly synthesised DNA denatured to single strands to allow for template for primers (95)

4- temp lowered again, new primers anneal (50-60) and cycle repeats

steps and temps of PCR

2^30 molecules

how many DNA molecules would you get from 30 PCR cycles starting with 1 DNA molecule

Kerry Mullis

who made PCR (and did a lot of acid)

sequence genome vs number of copies/molecules in sample

qualitative vs quantitative DNA sequencing

RNA is unstable, convert to more stable DNA before doing PCR

why use RT-PCR (reverse transcriptase PCR) when trying to sequence RNA

non-specific primers attach to 5’ and 3’ ends of strands

what are linkers and where do they attach to DNA

1. cut genome into many fragments

2. attach linkers to 3’ and 5’ ends and denature DNA (to single strands)

3. anneal to complementary primer on plate, add polymerase and bases to make complementary strand

4. complmentry strand is now attached to plate, dentaure and the other strand is washed away

5. DNA strand creates horseshoe by annealing to other primer on the plate, then complementry strand is made again.

6. Denature and now have double the DNA, and this is once again made into horseshoe and copied

7. process creates clusters of each fragment of DNA strand on plate

main steps of massive parallel sequencing

1. each nucleotided is tagged a differient colour

2. a new primer is added to the DNA strand

3. one dNTP binds, colour is recorded, flourescent tag (flourophore) is washed off allowing next tagged nucleotide to bind. Next nuclotide binds, colour recorded and then washed to allow for next.

4. Repeat until strand is sequenced.

from massive parallel sequencing how is DNA tagged and sequenced?

align cDNA with map, sequence by colour tags, read clone map to find mutation

steps to sequence fragments when have a DNA map and want to find mutation

random cDNA, sequence by colour tags, aligned randomly sequenced clones with computer

q

steps to sequence unknown genome (no map)

cDNA is the DNA reverse transcribed from a fragment of RNA, DNA is the entire DNA script

what is cDNA how is it different from DNA

1. RNA isolated and converted to cDNA by random primers and RNA dependant DNA pol

2. cDNA broken to peices

3. massive parallel sequencing used to sequence then align cDNA to genome

4. number of sequences that align to locus in genome are quantified and plotted

5. plot provides quantitative levels of transcription at each genome

Quantitative RNA sequencing (global analysis of genome)

uni-one peak (TATA/Inr)
bi- two peaks (GpC)

Uni vs bi directional transcription from eukaryotic promoter appearance on graph

immunoglobins

antibodies are _________ produced to combat exogenous protein

Ig

a special class of B lymphocytes rearrange ______ genes

antibodies

each B lymphocyte produces a unique antigen against exogenous protein, when invaded by antigen they produce a large amount of ______ to destroy it

monoclonal

antibody produced by one lymphocyte (one clone) good for a perscise target

polyclonal

many B-lymphocytes producing unique antigen targeting an antibody collectively are called _____ and are good for broad detection

inoculation of and animal and collection of its antiserum

antibodies can be collected and used in vaccines by

use specific antibody coupled to fluorescent dye to localize antigen in cell

immunoflourescence

antigen and antibody mixed with proteins and beads to form a complex with antigen. washing and seperating out the antigen, antibody, bead complex allows you to elute target protein (generally by western blot)

immunoprecipitation

to detect the specific binding location of a protien to DNA sequence (in vivo)

Chromatin immunoflourescence (ChIP) is useful for what

1. fix protein to DNA using membrane crosslinker formaldehyde

2. sonicate DNA into short fragments

3. add specific antibody (designed to target protein of interest)

4. purify to only segment with protein of interest

5. reverse formaldehyde cross linkage and isolate the DNA and subject to massive parallel sequencing and align random sequences by computer

ChIP steps

the pol 2 (and insulator) peak aligns with the start of the gene

what should you look for on ChIP graphs when trying to see if a gene is transcribed

CpG islands (but transcription eventually ends up only transcribing in the one open reading frame direction

type of promoter that initiates in two directions

TATA/ Inr

type of promoter that initiates in one direction

near the start site (start codon)

where are CpG islands in the DNA?

GTFs

in both CpG and TATA/ Inr what loads RNA pol onto promotor

TATA, Inr, BRE

what promotor elements are not found in CpG islands

70%

what percent vertabrate genes are transcribed by CpG island initiation

low, constant rate (essential genes for function replace worn out proteins)

what rate are CpG genes transcribed at

True, it is not defined

CpG transcription can initiate at any position? (T/F)

euchromatin

what type of chromatin is CpG found in

currently being made/ transcribed

define nascent

antisense stops, sense further elongates

after pausing what happens to sense and antisense direction of CpG initiated transcription

TF2D binds to TATA

what is first factor to bind when forming pre initiation complex (PIC), where does it bind?

TF2B and TF2A

what factors bind after TF2D?

TBP- TATA binding protien and TAF- TATA binding protien associated factors =TF2D

what are the components of TF2D (multiprotien complex)?

the RNA pol is recruited along with TF2F. The RNA pol unphosphorylated CTD interacts with the GTFs- forming the core PIC

once TF2D, TF2A, TF2B bind what happens next

TF2E and TF2H join. TF2H has kinase and helicase activity.

now that core PIC is formed what happens next?

opens the DNA helix using ATP and creates transcription bubble

what does TF2H helicase activity do?

phosphorylates (not to completion) the CTD to release the RNA pol from the promotor

what does the TF2H kinase activity do?

YSPTSPS

what is sequence of CTD?

EFs, elongation complex

what factors increase transcription, what complex is now formed?

5’ methyl guanosine cap

immediately after transcription begins what happens to nascent RNA

NELF and DSIF

After (partial) phosphorylation of CTD by TF2H kinase activity what negative elongation factors associate with RNA pol 2?

pause transcription downstream of initiation site, pausing allows for a check if transcription is occurring properly.

what do NELF and DSIF do? what is the purpose of doing so?

DSIF promotes pausing, however after complete CTD phosphorylation DSIF switches roles and helps with elongation

NELF helps to stabalize pausing, after complete phosphorylation of CTD NELF dissociates (and is replaced by PAF)

what is the difference between NELF and DSIF?

PTEFb (positive transcription EFb) kinase (aka CDka/CyCT kinase)

what enzyme completes the phosphorylation of CTD?

PAF, Spt16, DSIF (converted)

What are the positive elongation factors after complete phosphorylation of CTD?

PTEFb- kinase that fully phosphorylates teh CTD and NELF, triggering transcription to go ahead (past last checkpoint, can no longer stop)

what is another term for CDK9/CyCT? what does it do?

Retrovirus, uses reverse trascriptase to make inject its RNA into the hosts DNA.

what kind of virus is HIV, how does it insert genetic info?

euchromatin, more frequently transcribed and less dense- easier to access

what section of the chromatin does HIV generally aim for? why?

the 5’ end of the short HIV RNA

what part of the HIV RNA can be synthesized before the first pause?

TAR (trans activation response element)

what secondary stucture is created before the first pause by the HIV RNA

hold the polymerase in the paused position by inhibiting the PTEFb (CdK9/CyCT)

what does TAR secondary structure of HIV RNA do?

tat (trans activator of transcription)

what protein does HIV encode that binds to TAR to start transcription again?

when the affected cell (usually immune T-cell) is under stress polymerase at tat initiation site is released to produce tat

what triggers the production of tat?

PTEFb (phosporylates CTD and NELF releasing polymerase and transcribing the HIV virus)

what is activated when tat binds to TAR

throughout the DNA- more found in TATA than CpG, the least found in single celled eukaryotes

where are control regions (that proteins bind to) found in DNA?

proximal, distal

specific regulatory sequences- promoter proximal elements are _____ to start site and enhancers are ______ to start site

DNA binding domain, one or more activator/repressor domain

transcriptional activator proteins are modular and contain what domains?

Linker Scanning Reporter gene techniques

how can you find regulatory domains in DNA?

a gene that produces a detectable signal- used to monitor gene expression and find regulatory elements.

ex. tk- thymidine kinase can track enzyme production

ex. luciferase- can track light production

what is a reporter gene? give two examples:

on a plasmid that can be fused with gene/ promotor of interest

where are reporter genes found

False, the reporter reports based on transcription of gene of interest, uses gene of interests promoter

the open reading frame of the reporter plasmid has a promoter (T/F)

-upstream of reporter gene, clone large DNA chunk that you know controls gene of interest

-systemically replace small pieces (10bp) with linkers and produce many plasmids with linker subsitutions

-insert plasmids into cells, measure activity of the reporter

-if reporter loses activity you have scrambled the section with the regulator

how are reporter plasmids integrated into the gene of interest?

• open reading frame for enzyme to be used as indicator (such as kt or luciferase)

• no promotor and proximal regulatory sequences- closeby and tightly controlled

• multiple cloning sites (lots of restriction enzyme sites so you can insert into DNA fragments of interest)

• translation initiation elements- translate mRNA transcript into protein/ signal

• poly A signal downstream of reporter gene to ensure termination and the stability of reporter mRNA

key elements of reporter plasmids

a purposefully scrambled portion of the DNA to see if it is a region that affects transcription

what is a linker region

no transcription

if TATA box is scrambelled what do we expect to see from reporter?

reduced transcription

if a proximal enhancer is scrambled what can we expect to see from reporter?

both

are TATA regulatory elements up or downstream of start site?

proximal, distal, commonly found in introns (sometimes exons), can even be in adjacent genes (but not further than that)

how close to start site are TATA regulatory elements?

CpG islands

do CpG islands or single celled eukaryotes have a more complex system of regulators?

enhancers

______ can function over a long distance from start site (tens of kBPs)