MCB 317Genetics and Genomics

MCB 317 Topic 10, part 2,A Story of Transcription

Deletion andLinker Scanner Analysis

In vitro Txn Assay

Promoter not sufficient in vivo

Identification of Enhancers

Identify and define TBPand basal factors

Extract +Prom.-Enh.

Basal Facts. +Prom.-Enh.

Activated Txn(Enhanced) &Regulated Txn

Extract +Prom.-Enh.

ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter

“Activated” txn & Regulated txn

Identifying Proteins that Regulate Transcription

Experimental Continuum

• Biochemistry– Chemistry– Enzymology– Crystallography– Protein purification– Molecular biology

• Genetics– Population– Mutant screens– Classical e.g. Epistasis– Molecular genetics– Molecular biology

Fields-of-study function in conjunctionfew biologists/biochemists adhere to a particular sub-field

Genomics

Proteomics

“Factors” are required for specific txn of genes in vitro

gene gene

+

RNAPI, II or III

Radioactive nts+

+ RNAP

+ radiact. nts

+ Nuclear Extract

“Random” initiation Initiation at startSites and txn of specific genes

Conclusions

1. Cellular (nuclear) molecules other than the RNAPs mediate specificity of txn. Different than prokaryotes.

2. “Factors” recruit RNAPs to their Promoters!

How to Identify Txn “Factors”?

Controls allow interpretation of results.

One way to look at controls:

Positive control insures that Assay or experiment is working properly.

Negative control sets the “baseline” or defines the “signal-to-noise” ratio

Controls need to be includedin virtually all experiments

RNAPII Txn

• General transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

• General factors also called Basal Factors• Basal factors plus RNAPII sufficient for txn in vitro from a

gene and promoter• Required for txn of all RNAPII genes• Factors are (protein) complexes• Subunits: TFIIA = 2, TFIID ~ 15, TFIIE = 2, TFIIF = 3, TFIIH =

10 subunits• “Core” RNAPII = 12 subunits in yeast• Factors v. Fractions (Fxn)

Deletion andLinker Scanner Analysis

In vitro Txn Assay

Promotersufficient in vitro

Identification of Enhancers

Identify and define TBPand basal factors

Extract +Prom.-Enh.

Basal Facts. +Prom.-Enh.

Activated Txn(Enhanced) &Regulated Txn

Extract +Prom.-Enh.

ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter

“Activated” txn & Regulated txn

In vivo Txn AssayPromoter not Sufficient

What do factors do?

• Do they bind DNA?• Are they enzymes that modify the polymerase? • Do they bind RNAPII (ala Sigma Factor)?• Do they do any of a hundred other things?• Are they artifacts? [TFIIC, TFIIG]

Order of Addition Assay

How to identify where factors bind to genes?

1. Footprinting

2. Gel mobility shift assay (band shift assay)

Nucleases

• Site specific (restriction enzymes)• Exonucleases• DNAases• RNAases

“Footprinting”

DNAase I Footprinting

DNAase “nicks”one strand

Gel Mobility Shift = Band Shift= EMSA

(Electrophoretic Mobility Shift Assay)

“Native gels = Gels in which proteins remain functional

How to purify factors that bind DNA?How to assay for DNA binding proteins?

1. Footprinting2. Gel mobility shift assay = Band shift assay3. Affinity purification

Bandshift = simple assay to monitorpurification

Assay fractionsfor factors by band shift gel

Gel Mobility Shift Assay = Band Shift Assay

“Free” DNA = unbound DNA; only small % needs to be bound

Why is small amount of DNA shifted experimentally significant?

How does this compare to footprinting?

Combine gel shift and Footprinting

DNAase -> Run Gel -> cut out shifted bands -> denature -> sequencing gel

Lodish 11-13

Affinity purification of DNA binding proteins

Which txn proteins bind DNA?

• General txn factors?

• Activators?

“Real” Data: actual basal factors, not hypothetical fxns

Therefore, TFIID must contain additional components that are required for activated

levels of txn

TAFs = TBP Associated Factors

Results without Experiment

TFIID is a complex of ~15 proteins:TBP plus 14 TAFs

TBP is at the heart of RNAPIIPromoter function:

it binds TATAit binds TAFsit binds basal factors

A current model for assembly of the general txn factors at promoters

Does this process actually occur in vivo?

Remember, we’ll talk about it later

TFIIH = Helicase and RNAPII CTD kinase

How would you generate the data for a step-wise assembly model?

Stable pre-initiation Complex (PIC)

TFIIH

Title

“Complex” binds multiple sites Cooperativity: strong sites help binding at weak sites Order of binding- is TBP/TFIID always first?

How are txn by RNAP I, RNAP II and RNAP III similar and dissimilar?

RNAP I Txn

RNAP III Txn

Deletion andLinker Scanner Analysis

In vitro Txn Assay

Promotersufficient in vitro

Identification of Enhancers

Identify and define TBPand basal factors

Extract +Prom.-Enh.

Basal Facts. +Prom.-Enh.

Activated Txn(Enhanced) &Regulated Txn

Extract +Prom.-Enh.

ActivatorsCo-activators + Enhancer &TBP & TAFs Promoter

“Activated” txn & Regulated txn

In vivo Txn AssayPromoter not Sufficient