Transcription regulation

Transcription initiation is regulated by

proteins (trans factors) that bind to specific sites (cis elements) on the DNA

Transcriptional regulators can be both

positive and negative

activators are trans acting factors upstream of promoter

repressors are trans acting and bind to DNA to block promotor

polycistronic

in bacteria groups of related genes are grouped together under shared promoter = operon

makes one polycistronic mRNA

genes are co-regulated

anti-sigma factor

binds to alternate sigma factor and blocks binding to promotor

how small molecules can regulate transcription

bind to transcription factors

ex: tryptophan binds to repressor so no more tryptophan can be made

Lac operon regulation

When lactose is absent Lac repressor binds to Lac operator

When glucose is absent CAP activator can bind to promoter

transcription only occurs when glucose is absent and lactose is present

cyclic AMP also needed for CAP to work

Attenuation

Regulation of transcription termination in bacteria

low levels of tryptophan, ribosome pauses at Trp codons in leader peptide upstream fo Trp operon

stem loop 2:3 is formed blocking pairing of sequence 3 and 4 to form intrinsic terminator

high levels of tryptophan ribosome doesnt pause and covers region 2 so 3:4 stem loop can form intrinsic terminator → polymerase dissociates and does not make more trp

how can we compare RNA levels in cells treated under different conditions?

RNA-seq

to compare between samples need to normalize read count for a gene to the total reads in the experiement

RPKM and FPKM

reads per kilobase per million

fragments per kilobase per million

what does this show?

RNA seq heat map

each line is a specific gene

each column is a condition

compares changes in gene expression across many genes

if similar pattern in genes then may be coregulated

enhancers

eukaryotic regulatory regions that can bind far away from gene being regulated

upstream or downstream

loop DNA so regulatory elements contact Mediator

can have multiple enhancers for one gene

Co-activators and Co-repressors

bind to DNA binding proteins

do not bind directly to DNA

How does Gal4 recruit RNA polymerase?

through a co-activator complex

in presence of galactose Gal3 binds to Gal80 and exports it from the nucleus so it can’t inhibit Gal4

then Gal4 recruits SAGA co-activator complex which promotes assembly of mediator complex

histone modifications and transcription

H3K9me3 and H3K9Ac = gene expression

H3K9me3 = gene silencing and heterochromatic formation

H3K27me3 = gene silencing

specific modifications found at certain locations relative to gene body

How to map histone modifications

ChIP-seq

histone deacetylase

represses transciption

spreading of histone modification by sequential action of modification enzymes

ORC, Rap1 and ABF1 recruits Sir1 to Rap1 then Sir 2, 3, 4 bind and Sir2 deacetylates H3 then sir 3 and 4 do the same which brings in sir2 again

Northern blots

examine levels of a single specific RNA similar to southern blot

to amplify mRNA need to convert to cDNA and add adaptors for PCR primers to bind

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

determines relative amount of an RNA in diff samples

number of cycles it takes to cross threshold is proportional to amount of input DNA

Single cell RNA-Seq (scRNA-seq)

used to identify cells that express similar set of genes