BIMM final real

different types of RNA polymerase

RNA polymerase I: pre-rRNA

RNA polymerase II: mRNA, snRNA, siRNA, miRNA

RNA polymerase III: tRNA and other small stable RNAs

all have 2 b subunits, 2a, and 1 w

c-terminal domain (CTD)

a 7 amino acid repeat on RNA pol II. Phosphorylation of CTD is critical for transcription initiation and elongation.

conserved sequence

the sequence in the gene core promoter that allows RNA pol to interact with the DNA by providing a place where required proteins bind.

lies close and upstream to +1, around 20 or 35 basepairs

This is typically the TATA box and TATA binding proteins (TBP).

TFIID

general RNA pol II transcription factor made of TBP and TBP associated factors (TAFs). — tata box binding

TFIIB

bridges RNA pol and TFIID

transcription pre-imitation complex (PIC)

RNA pol-II with its transcription factors

TFIIE

involved with DNA melting

TFIIH

has DNA helices and pol II CTD kinase function

Promoter-reporter gene fusion

using recombinant DNA to have a promoter code a reporter gene. Then observing the color to determine how active promoter is

Electrophoretic mobility shift assay (EMSA)

running dna and proteins in a gel, if the proteins bind, then the band will be higher

linker scanning

systematically deleting sequences in the promoter to identify essential sequences for transcription.

transcription

production of a primary RNA transcript from a gene

RNA polymerase

requires a ssDNA template strand and rNTPs. works by using the 3’ OH to polymerize the alpha phosphate on the incoming rNTP

steps of transcription

1. initiation (promoter recognition and binding, DNA melting, RNA synthesis starts

2. elongation (RNA synthesis)

3. termination (release of RNA pol and primary RNA transcript)

Transcription initiation

1. binding

2. melting

3. putting down 2 bases

northern blot

just like southern blot but with RNA molecules instead of DNA. this way can identity if a gene was transcribed into RNA or not.

core promoter regions

This is a specific region of DNA (typically about 50 to 100 base pairs long) located right around the transcription start site (TSS) of a gene that dictates transcription start site (+1) and direction of transcription.

It acts as the primary docking and assembly platform where RNA polymerase and general transcription factors bind to start transcribing a gene.

it is required for transcription.

sigma factor

in bacteria, RNA poly interacts with sigma factor that bind to conserved sequence motifs in core promoter regions

transcription rate regulation types

global transcriptional regulation (transcriptional co-regulation of multiple genes for example in response to heat shock)

ex) regulating sigma factors

gene specific transcription regulation (transcriptional regulation of specific genes)

ex) regulation of lac operon

global transcription regulation

accomplish this by regulating the availability/activity of specific sigma factors.

gene specific transcription regulation example (LAC operon)

LAC operon allows for the cell uptake and breakdown of lactose into glucose

in low/no lactose environments, lac repressor binds to lac operator and prevents transcription.

in high lactose conditions, the repressor dissociates from the operator.

in low glucose, the CAP binds (after cAMP binds to it) to upstream primer and activates transcription.

high glucose, CAP dissociates.

this way lactose in metabolized only is glucose is not available

how glucose regulates lac operon through CAP

core elements found in eukaryoktic promoters

1. Tata box

2. TFIIB recognition element (TRE)

3. Initiator element

4. downstream prmomoter element

5. CpG island

DNA elements controlling RNA pol II transcription

core promoter: dictates start site and direction of transcription

promoter proximal elements: enhances or represses, positioned -1 to -200 (~6bp)

enhancers or silencers: can be 1000s of nucleotides upstream or downstream of promoter (~200bp)

domains of transcription factors

specific binding of TF

• major grooves

• interaction with bases give specificity and interaction with backbone gives affinity

TF regulation in transcription initiation

• pioneer TF can bind to condensed chromatin and decondense it and expose all the dna for transcription

• can also stabilize or destabilize the PIC

enhancers

• distant DNA elements that can activate or repress transcription and is distance and orientation independent

• linearly distant but comes closer in folding

TF expression in only some cells

?

• if the chromatin is decondensed or not

• is the co factor is present