Bio 23 Class Notes July 17, 2001

Initiation — Most gene regulation is at this step

Elongation

Termination

Initiation elements

1. Cis acting elements
1. DNA sequences required for transcription initiation
2. Trans acting elements
1. Protein factors bind to DNA or other protein factors

2 Main Types of Cis Acting Elements (see Figure 20.1 in Lewin)

1. Close to transcription start site.
2. Spans about 200 base pairs upstream of transcription start site.
3. Specifies location of transcription start site.
4. Supports transcription at normal levels
1. Enhancers enhance levels of transcription
5. More variety in sequence than prokaryotic.
6. Promoters are Modular
1. Multiple sequence elements in one promoter.
2. Bind multiple protein factors.
7. Elements that are about 50 bp upstream are fixed in postion and orientation.
8. Other regions can be moved around.
9. Need promoters to get transcription to get transcription from any gene.

1. Enhancers
1. Not necessary for every gene
2. Mainly used for RNApol II genes (few for RNApol II & III).
3. Can be very far from transcription start site (several kb).
4. Have multiple sequence elements closely clustered.
5. Important for tissue specific and temporal regulation.

Transcription factors required for initiation that are not a part of RNA polymerase.

Three Types:

1. General
2. Upstream
3. Inducible

1. Act on 50 bp region of promoter.
2. Required for initiation or transcription.
3. Each RNA polymerase has it’s own set of general transcription factors.

General transcription factors + RNA polymerase is called the Basal Transcription Apparatus.

1. Transcription factors (other than general) that bind upstream of tx start site.
2. Ubiquitous — Always present in all cells. They don’t give tissue specific regulation.
3. Can be bound to promoters or enhancers.

1. Turned on at specific time or place. Or turned on in response to a signal
2. Function like upstream factors but have regulatory role.
3. Can be synthesized or activated.
4. Regulate expression in regards to time and space.
5. Can respond to environment changes (stress).
6. Mainly positive control.

Most eukaryotic genes are under positive control. They are OFF unless turned ON.

To talk about regulation you must define the regulatory DNA sequences and identify the proteins involved.

To study the DNA sequences you can cut and join pieces of DNA and assay for transcription.

To assay:

1. Look for mRNA by hybridization
2. Assay for gene product (like we did in lab).

In this way you can determine what regions of the promoter sequence are necessary for transcription.

You must keep the length of the promoter constant as you cut it apart. To do this you use filler DNA.

(See figure 20.3 in Lewin)

4 Assay Techniques

1. Xenopus Oocyte (big cell, frog egg):

Inject DNA into nucleus and see if transcription occurs. Easy in-vivo assay but you cannot control proteins present.

2. Transfection of tissue (cultured cells)

DNA gets into cell by electroporation or salt wash. Advantage is that you can control the cell type.

3. Transgenic Animals.

Insert DNA into the genome of a germ line cell. All offspring will have gene. Good for studying developmental regulation but it’s hard to control the position of insertion of gene (due to chromatin).

4. In-vitro assay

Mutation analysis can also be used to see if particular mutations increase or decrease transcription. Cross-linking experiments can also tell who’s touching who in the complex.

There are 3 types of RNA polymerases in Eukaryotes.

1. RNAP I — rRNA (28s, 18s, 5.8 s)
2. RNAP II — mRNA, small nuclear RNA (snRNAs which are part of snRNPs)
3. RNAP III — tRNA, small nuclear RNAs, 5s rRNA

All 3 are large complexes (~500 kD) and made of 8-14 subunits.

RNAP I (See Figure 20.4 in Lewin)

1. Makes many identical copies of rDNA (millions of ribosomes).
2. All rDNA genes have a bipartite promoter (2 parts)
3. Fixed promoter, 2 parts.
1. Core promoter.
1. Close to transcription start site. —45 to +20
2. UBF1 (Upstream Binding Factor) binds and recruits SL1
1. SL1 contains 4 proteins. One is TBP: — TATA Binding Protein
1. TBP binds to Pol I, II, and III promoters but does different things at each. At RNAP I it doesn’t bind directly to DNA but does help form a surface that RNAP I recognizes
2. Upstream Control Element
1. Further upstream from transcription start. —180 to -107
2. Also binds UBF1 and SL1
4. UBF1 and SL1 recruit RNAP I and determine where transcription starts.

RNAP III (See Figure 20.7 in Lewin)

Has well defined promoters

1. Upstream Promoter (See Figure 20.6)
1. Upstream of transcription start site
2. Used for snRNA
3. Recruits TFIIIB (a multi-protein complex)
1. The TBP does recognize the TATA box and helps recruit RNAP III.
2. Internal Promoter
1. Downstream of transcription start site
2. Used for 5S rRNA and tRNAs
3. There are 2 types of internal promoters. Each have 2 internal binding sites
1. The A-C complex recruits TFIIIB which recruits RNAP III
1. TBP does not interact with DNA
2. The C complex recruits TFIIB which recruits RNAP III to start site

Allows cells to express different gene products.

1. Genes transcribed by RNAP II respond to environmental and developmental cues
2. Changes in gene expression lead to:
1. Metabolism adjustment
2. Cell division.
3. Changes in cell fate.
4. Programmed cell death
3. Enhancers usually responsible for response to cues
4. The GC box and CAAT box determine efficiency of transcription. They have variable distance and orientation.
5. The TATA box controls transcription start site. Orientation and position are not variable.
6. RNAP II needs GTF to form basal transcription apparatus.

1. Assembly of initiation complex. TFIID, A, B, F
2. ATP dependent formation of open complex. TFIIE, H
3. Promoter clearance. TFIIH, (F)

1. TFIID Binds first.
1. TBP is always present in TFIID
2. TFIID also contains TAF's (TBP associated factors) TAFs can vary for different promoters there are others (TAF’s)
3. TBP binds TATA Box in minor groove
4. TBP bends the DNA 80 to 90 degrees.
5. One surface of TBP interacts with DNA and the other with TAF’s and GTF’s
2. TFIIA, B bind after IID and stabilize the complex.
1. Interact with TBP and DNA
3. TFIIF required for RNAPII to bind to promoter
1. Interacts with RNAPII before it binds to complex
2. IIF has 2 subunits. One has helicase activity and one is sigma homolog.
4. When RNAPII docks, C terminal tail (CTD) of the large subunit interacts with TBP and TAFs

Open Complex formation.

1. TFIIH has 3 activities
1. ATPase
2. Helicase
3. Kinase- Phosphorylates CTD which is important for promoter clearance.

Transcription then begins

1. One more rate limiting step in initiation is promoter clearance. The helicase activity of IIH (maybe IIF) and kinase activity of IIH are required for RNAP II to move away from the promoter.
2. CTD has many 7 a.a. repeats and can be phosphorylated many times. Phosphorylation by TFIIH is required for promoter clearance.
3. TFIIH travels with RNAPII and is required for elongation.