MOLECULAR BIOLOGY, LECTURE 10, Oct. 10, 2000

EUKARYOTIC TRANSCRIPTION

I. Each eukaryotic cell contains 3 RNA Polymerases (p266-281).

1. Initially recognized as distinct proteins (Fig. 10.2) by column chromatography

2. Display differential sensitivity to a-aminitin (Fig. 10.6, 10.10)

3. Catalyzes transcription of different classes of RNAs (Fig10.7, 10.8, and 10.11)

A.RNA polymerase I - synthesizes large rRNAs (28S, 18S, 5.8S)

B.RNA polymerase II - synthesizes mRNAs & most small nuclear RNAs

C.RNA polymerase III - synthesizes low MW RNAs (various tRNAs & 5S rRNA)

4. Composition: (Yeast Pol II). Purification and genetic analysis in yeast. (Table 10.1, 2)

3 core subunits, RBP1, 2, 3, homologous to E. coli b', b, and a subunits

5 comon subnuits, shared by all three yeast Pol: RBP5, 6, 8, 10, 12.

2 are nonessential: RBP4, 9

RBP7 and RBP11 as a separate group

5. CTD of the Pol II, consisting of multiple repeats of a consensus sequence of

7 amino acids (YSPSTPS) that can be highly phosphorylated.

It is thought this domain plays an important role in various RNA processing events.

6. The structure of RNA Pol II: a moving PolII

II. Regulatory Sequences (p281-289, p123-127)

    1. Experimental techniques to characterize regulatory sequences

Gel mobility shift assay (Fig. 5.31)

DNA footprinting (Fig. 5.32)

Functional assay: (Deletion, Linker scanning, site-directed mutagenesis)

2. Pol II Promoters (Fig 10.25),

A. TATA Box( Fig. 10.26-28):

positioning of the start of transcription

in some cases, required for promoter activity (Fig. 10.29, 30)

C. Initiator/Downstream element:

I: PyPyANTPyPy; DSE; no consensus sequence

B. Upstream element

GC Box, orientation-independent, but position-dependent, SP1

CCAAT Box: CTF and C/EBP

3. Enhancers/silencers/insulators

position and orientation-independent (the 72 bp repeat of the SV40 early gene)

E: interact with GTFs and RNA Pol to assist the formation of a preinitiation complex

S: block the formation of basal transcription apparatus

The same element can have both activities, depends on the binding factor

Insulators block actions of either E or S

III. General transcription factors and assembly of preinitiation complex (Pg 300-323).

    1. purified Pol II can not initiate transcription on a dsDNA template
    2. step-wise assembly of an pre-initiation complex in vitro. (Fig. 11.3, 11.6, 11.31)

      3. A.TFIID recognizes and binds to the TATA box

      B.TFIIA binds and stabilizes TFIID binding.

      C.TFIIB binds to TFIID and spans ~30bp to determine the transcription start site.

      D.TFIIF recruits RNA Pol II into the pre-initiation complex by binding to TFIIB.

      E. Finally, TFIIE and TFIIH are bound to complete the pre-initiation complex.

    3. Features and function of individual GTFs:
    1. TFIID is a complex containing a TATA-box binding protein (TBP) and 8-12 TBP-associated factors (TAFIIs; Fig. 11.12)

      2. TBP, a saddle-like structure, binds to the minor groove to bend DNA. And supports basal trannscription.

      Function of TAFIIs: binding to core promoter (Fig.11.17a, b)

      Interact with gene-specific activators (Fig.11.17c)

      Provide enzyme activities: HAT and kinase

      Form a nucleosome–like structure?

    2. TFIIA is composed 2 subunits and function to stabilizes TBP/TATA-Box interaction.
    3. TFIIB:is a single-subunit factor, recruits RNA PolII-TFIIF to the PIC and is involved in start site selection.

A model of TFIIA-TFIIB-TBP-TATA box complex

C. TFIIF consists of two subunits and is responsible for docking the PolII to the PIC.

D. TFIIE/TFIIH (Fig11.25, Fig.11.31)

TFIIE is a heterotetramer and is required for recruitment of TFIIH

TFIIH is a 9-subunit protein complex and plays multiple roles
    1. The ATPase/helicase activity is essential for promoter opening
    2. A kinase polymerase at CTD which allows the POLII to shift from initiation to elongation
    3. Many subunits of TFIIH are involved in the transcription-linked repair of DNA damage

A link to yeast RNA PolII and its general transcriptional factors:

A link to animated models of RNA PolII transcription initiation complex
    1. RNA Pol II Holoenzyme

It is composed PolII, TFIIB, TFIIF, TFIIH, Srb-Med, Srb10-CDK, and Swi-Snf complexes.

SRB proteins were identified by mutations that suppress the cold-sensitive phenotype of deletions in the CTD of the large subunit of RNA polymerase II and were shown to form a high molecular weight complex with RNA Pol II (about 1200 kD)

MED proteins were identified biochemically as a protein complex required for the response to activator proteins

Srb10-CDK is a complex composed of 4 SRB proteins that has a CTD kinase activity. It plays a negative role in transcription.

Swi-snf complexes are implicated in transcriptional regulation by chromatin remodeling..

Direct recruitment of the RNA PolII holoenzyme.

The holoenzyme is thought to bind to promoters (as directed by TFIID or TBP) in response to activator proteins