Biochem 508 Lec 9: Regulation of Gene Expression II

What eukaryotic enzyme is responsible for the synthesis of mRNA?

Eukaryotic RNA Pol II

Prokaryotes have a __ RNA Pol

Single

Eukaryotes have ___ RNA Pols, to make different kinds of RNA

multiple

Can the eukaryotic RNA Pol II bind to the DNA template directly on its own?

NO! Needs protein cofactors to bind, UNLIKE the prokaryotic RNAP, which can bind directly but needs sigma factors for specificity

Are eukaryotic genes organized into operons like bacteria?

No! In general, eukaryotic genes are each under their own promoter

What is DNA bound to histones and other proteins called?

Chromatin

What are the two states of chromatin we should know?

• Euchromatin: less condensed, available for transcription

• Heterochromatin: more condensed, transcriptionally repressed

How many subunits are histone proteins made of? What are they named? What kind of supercoiling do they induce?

• 8

• H2A, H2B, H3 and H4

• Negative toroidal

H1 linker proteins are how we …. and they are also modified post-____.

Organize nucleosomes into condensed chromatin, post-translational

H1 sticks to nucleosomes and facilitates interactions between them

Both chromatin ___ and ___ can affect levels of transcription.

structure (euchromatin or heterochromatin), remodeling

What are 3 examples of post translational histone modifications?

• de/phosphorylation (only of H1)

• de/acetylation (Lys)

• de/methylation (Lys)

Remodeling of nucleosomes requires ___.

ATP

What is the role of SWI/SNF (yeast) or BAF (humans)? It is a large ___ of proteins.

• Shuffle nucleosomes around to let RNAP access genes. Can take the DNA out of the nucleosome, move the histone, eject the histone, etc

• Complex

Is adding acetyl groups to histones activating or deactivating? What enzyme can add acetyl groups?

• Activating!

• Histone acetyl transferases (HATs)

What enzyme can remove acetyl groups? Is this activating or deactivating?

• Histone deacetylases (HDACs)

• Deactivating

More condensed proteins have ___ levels of histone H1

Higher- H1 links nucleosomes together

RNAP can have a direct effect on chromatin ___.

Condensation

How might RNAP directly affect chromatin structure?

• RNAP association with elongation factors allow RNAP to move through nucleosomes by prying the DNA off the histone cores

• But, if RNAP stalls on compact chromatin, chromatin modifying enzyme are recruited to decondense the chromatin, allowing RNAP to continue

What is a special feature of RNAP II that the other eukaryotic RNAPs don’t have?

Long C-terminal tail that is the target of phosphorylation

What are the proteins called that are needed in ALL cells for transcription initiation?

General/basal transcription factors

Basal RNAP II transcription factors (TFIIs) assemble with ___ to form the ….

RNAPII, transcription pre-initiation complex

TFII’s and the PIC they form open a ….

transcription bubble for RNA synthesis

What is TFIID?

TATA Binding protein

Recognizes the TATA Box

RNAP II is really a complex of __ protein subunits

12

Binding of TFIID to the TATA box …

Kinks the DNA template, and might attract other proteins to the promoter

In eukaryotes, the TATA box is the -___ consensus seq

-30

Name the order of binding of the TFIIs

1. TFIID (TATA binding protein)

2. TFIIB (recruits Pol II)

3. TFIIA

4. TFIIF

5. TFIIE

6. TFIIH

Which TFII is responsible for opening the transcription bubble at the initiation site?

TFII H

TFIIH is an …

ATP dependent helicase

What modification does TFIIH make to RNAP?

Phosphorylates its C terminal tail

Describe the peptide seq of the RNAP C terminal tail

Repetitive, rich is S, T and Y

What is the significance of different RNAP C terminal tail patterns?

Indicates different stages of transcription and which recruits different sets of enzymes and co-factors to the polymerase

Write out how all the TFIIs bind

1. TFIID (TATA Binding), bends DNA near promoter

2. TFIIA and TFIIB join

3. TFIIB recruits RNAP II

4. TFIIE, TFIIF, TFIIH join the complex

5. TFIIH hydrolyzes ATP to open the DNA near the initiation site

6. RNAP carries out abortive transcription

7. **TFIIH phosphorylates the RNAP C terminal tail

8. RNAP II escapes the promoter and begins processive elongation of mRNA

What are transactivators

Sequence specific DNA binding proteins that promote RNAP initiation at promoter sites

Transactivators bind to…

cis acting regulatory sequences

Transactivators have separate….

DNA biding domains and activating domains

What protein-protein interaction motif is important for the formation of DNA binding protein complexes?

Leucine zipper

Fos and Jun are…

Members of a family of related transcription factors that dimerize via a leucine zipper structure

Leu residues are spaced every __ AAs in a leucine zipper

7

Alpha helices have __ AAs residues per turn

3.6

Homo vs hetero oligomerization of gene regulatory subunits

What is the significance of this?

• Homo: the same TF binds to the promoter

• Hetero: Multiple TFs bind to the promoter

Provides a way to increase the number and diversity of genes expressed under various conditions from a limited number of TFs

Do co-activators bind to DNA directly?

No! They facilitate protein-protein interactions

Co-activator transcription factors act as a …

Bridge between DNA-binding proteins and the RNAPII pre-initiation complex

What is the mediator complex and what are its functions

• A major co-activator of RNAP-II dependent transcription

• Functions in the recruitment and stabilization of RNAPII

• Interacts with DNA binding activators and RNAP II C-terminal tail, stabilizes TFIIH phosphorylation of RNAPII C-terminal tail

Co-repressors __ regulate transcription

negatively

Co-repressors recruit ____, and compete with ___

Histone deacetylases, co-activators