What is the difference between basal and activated transcription?

Regulation of TranscriptionI. Basal vs. activated transcription for

mRNA genesA. General transcription factor (TF) vs. promoter-specific

1. general TFs are required by all mRNA genesa. an absolute requirementb. transcription can occur alone with these

factors is by definition the basal level of transcription2. promoter-specific TFs are different for each gene3. the promoter-specific TFs are required for

maximal level of transcription or for activated transcription (induction)B. a third state is that of a repressed state

Regulation of Transcription

What is the role of the response elements and associated factors

How Can You Test the Functional Activity of an

Activation Domain?

Targets of Activation Domains

• General Transcription Factors– TBP (TFIID)– TFIIB– TFIIA

• RNA polymerase II• Coactivators

– Mediator– TFIID

Role of Activation Domains

1. Recruitment• Bind Faster• Remain bound longer

2. Conformational change• Active vs. inactive configuration• Blocked vs. accessible

3. Covalent modification

Coactivator versus Repressor (corepressor)

Activation and Repression of Transcription

Regulation of TranscriptionII. Question of Activation

A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors

induction: heat shock, heavy metals, viral infection, growth factors, steroids

4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins

1. response elements shared2. example of homeodomains

Regulation of TranscriptionII. Question of Activation

A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors

induction: heat shock, heavy metals, viral infection, growth factors, steroids

4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins

1. response elements shared2. example of homeodomains

C. regulating the activity of the transcription factors

What are the DNA binding domains of the TFs?

DNA binding domainsA. Zinc fingers

1. Cys2-His2 fingers: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X-Hisa. example is TFIIIA has 9 Zn finger repeatb. typically the number of fingers range from 2-9c. can be involved in binding to RNAd. not all Zn fingers are used to bind DNA, nor are they always

part of a transcription factor2. Cys2-Cys2 fingers: Cys-X2-Cys-X13-Cys-X2-Cys

a. found in steroid receptorsb. typically nonrepetitivec. binding sites are short palindromesd. bind as dimers

3. Binuclear Cys6 finger: Gal4 DNA binding domains

DNA binding domains

B. Steroid receptors

DNA binding domainsB. Steroid receptors

1. Ligand mediated activation2. Functional Domains

a. DNA bindingb. ligand binding - hormonec. activation domain

3. Two classesa. form homodimers: bind consensus half site (TGTTCT, except for ER is TGACCT)b. form heterodimers: bind half sites of

TGACCT, direct repeatsc. spacing of the half sites is crucial for the

degree of specificity

DNA binding domainsC. Leucine zippers - dimer formation

1. brings 2 DNA binding domainsin close juxtaposition

example is Gal4 2. amphipathic alpha helices with

Leu residues on one faceLeu repeats every 7 amino acid

3. interface forms a coiled coil

DNA binding domainsD. bZIP example is GCN5

1. basic region attached to a leucine zipper2. is a dimer kept together by the leucine

zipper3. an alpha helic containing basic residues

contacts the major groove of DNA4. contacts are made with the portion of the

bases exposed in the major groove and some phosphate backbone contacts

DNA binding domains

E. bHLH domain1. basic helix loop helix motif2. positively charged alpha helix binds

to major groove3. two other alpha helices form a four

helix bundle in dimer4. many will also contain a leucine

zipper

DNA binding domains

Dimer formation regulates the activity of the transcription factor

Activation Domains

A. Acidic activators - example of Gal4pB. Glutamine rich domainC. Proline rich domain

Transcription ElongationA. General

1. in vivo rates are 1200-2000 nucleotides/min

2. in vitro rates are 100-300 nucleotides/min3. elongation is not a monotonic continuous

processa. there are strong pause sitesb. effects of chromatin on process

4. pausing versus arrest (definition of)

Transcription ElongationB. Negative elongation factors (N-TEFs)

1. DSIF2. factor 2

C. Positive elongation factors (P-TEFs)1. prevent sequence dependent arrest (i.e. TFIIS or SII)

nucleolytic cleavage/ backtracking2. catalytic activity (TFIIF, elongin, ELL complex)3. regulates the rate of elongation

through chromatin (FACT)