Transcription in Eukaryotes

CTD (C-terminal domain)

eukaryotic region that is involved in a LOT of things. A docking platform for other factors involved in transcription

Pol I RNA Products

rRNAs

Pol II RNA products

mRNA

Pol III RNA products

tRNA

Pol I promoter elements

• UCE

• core sequence

Pol II promoter elements

• BRE

• TATA box

• Inr

• DPE

Pol III promoter elements

• Box A, Box B

• Box A, Box C

• TATA box

TATA box characteristics

• sequence that contains TATAAA near position -30

• common sequence in eukaryotic promoters

• not all promoters have TATA boxes

which promoter(s) doesn’t carry a TATA box

Pol I

TBP- TATA binding protein

• binds to the TATA box

• involved in transcription initiation of almost all genes (even ones that lack a TATA box)

TAFs - TBP associated factors

• help recruit associated factors

Pol I Promotor characteristics

• contains core sequence and upstream control element (UCE) → sits around promoter

• UBF (upstream binding factor)

  • binds to core sequence and UCE

• SL1 complex (protein)

  • contains TBP and TAFs

  • connects UBF and helps to recruit RNA Pol I

Pol III Promoter characteristics

• all their targets are short (less than 300 nucleotides) and untranslated

• part of their promoter is within the gene (downstream of the start site)

• all Pol III promoter require TBP binding to a TATA box, with a complex called TFIIIB (TF3B) → has the TATA box

• all have TFIIIC (“TF3C”) binding to boxes downstream of the start site

• TFIIIC gets kicked off by Pol III once transcription begins

Pol II promoter sequences

• Inr-initiator, at the start site

• DPE- downstream promoter element

• TATA box- binds TBP

• BRE- TFIIB Recognition Element, bind TFIIB (BRE recruits TFIIB)

Pol II Transcription Factors

• TBP- Binds TATA box

• TFIIB- binds BRE

• TFIID- massive complex with many TAFs, and TBP

preinitiation complex converts to initiation complex

leads to

1. loading of DNA into entry channel

2. melting of DNA

3. formation of transcription bubble

eukaryotic transcription

• phosphorylation of CTD region DURING INITIATION is involved in disengaging from the promotor (goes from giant pre-initiation complex to full initiation complex)

• elongation similar to bacteria but facilitated by elongation factors (8-10bp)

• during termination, CTD is dephosphorylated

transcription termination (Pol II)

Torpedo model:

1. Termination sequence AAUAAA signals for a complex (CPSF) to cleave mRNA. the 3’ end is further processed

2. transcript is finished, Pol II continues to transcribe

3. Xrn2 (an exonuclease, starts from the outside then cuts in) begins to chew up the leftover RNA

4. when it catches up to Poll II, it removed the transcript and ends transcription

   • similar to Rho-dependent termination in bacteria

what are ways to measure gene expression?

• Norther blot

• RT-PCR

• qRT-PCR

• RNA sequencing

Northern blot

used to analyze RNA amounts and sizes

Steps of northern bloting

1. RNA extract is run through and agarose gel

2. The RNA is then transferred from the gel to a nitrocellulose membrane

3. The membrane can be probed with a DNA probe that is radio labeled

   • probe will bind to complementary RNA sequences

4. the membrane is imaged with x-ray film to detect the presence of the radio labeled probe

   • The intensity of the band is proportional to how much RNA is present

PCR- Polymerase Chain Reaction

amplify short regions of DNA in order to visualize it

uses 2 primers

each cycle “doubles” the amount of product

RT-PCR (Reverse Transcriptase PCR)

a way to convert RNA to DNA in order to amplify using PCR

visualizes RNA transcripts without using a Northern blot

Steps of RT-PCR

1. treat RNA with reverse transcriptase enzyme to convert to cDNA (complementary DNA)

2. use cDNA in a PCR reaction

Quantitive PCR (or qPCR)

method to quantify the amount of PCR product and thereby the amount of starting DNA (removed the reason to run gels)

how does quantitative PCR work?

• there is a dye that binds to DNA used in a PCR reaction

• the dye gives off a fluorescent signal which is proportional to the amount of DNA in the tube

characteristics of SYBR green

• binds only to double strand DNA (dsDNA)

• when bound, it emits fluorescent signal

• the signal is proportional to amount of double stranded DNA

• in PCR reaction, can measure signal after each cycle

• signal should double each cycle as amount of dsDNA doubles

qRT-PCR

qPCR can be used to measure gene expression if RNA is converted to cDNA then analyzed via qPCR

loading control

primer for a gene whose expression is likely to be the same in every cell.

used to normalize all your samples to one another

experimental control

a sample to compare your sample of interest to

NextGen Sequencing aka High Throughout Sequencing

a method to sequence massive amounts of DNA at once

how NextGen sequencing works

sequences short regions of DNA but at a massive number and assembles them via computer

• RNA is not directly sequenced this way. Instead it is prepared into cDNA libraries or RNA-sequecning (RNA-seq)