Methods for StudyingEukaryotic Gene Control

1. Multiple eukaryotic RNA polymerasesfor rRNA, tRNA and mRNA synthesis

2. Methods for Mapping cis- regulatory sequences: proximal and distal elements

3. Finding the Start of Transcription

4. Complex Cis-control Modules are Bound by sequence specific factors to direct temporal and spatial regulation

Assay for non-specific RNA polymerase activity

Non-specific or syntheticDNA fragments

+ RNA polymerase

+ [a-32P]rNTPs/ Mg+2

RNA* **

+ [a-32P]rNTPs

Trichloroacetic acid (TCA) insoluble TCA soluble

The separation and identification of the three eukaryotic RNApolymerases by column chromatography.

A protein extract from the nuclei of culturedfrog cells was passed through a DEAESephadex column. Adsorbed proteins wereeluted (black curve) with a solution ofconstantly increasing NaCl concentration.

Fractions containing the eluted proteinswere assayed for the ability to transcribeDNA (red curve) in the presence of the fourrNTPs.

The synthesis of RNA by each fraction inthe presence of 1 mg/ml of a-amanitin alsowas measured (blue curve).

Three types of RNA polymerase in eukaryotic nuclei

RNAP Location RNA synthesized a-amanitin

I Nucleolus Pre-rRNA, except 5S InsensitiveII Nucleus Pre-mRNA, some snRNAs Sensitive to 1 mg/mlIII Nucleus Pre-tRNAs, 5S RNA,snRNAs Sensitive to 10 mg/ml

Pol I: single primary transcript, ~200 copiesPol II: ~30,000 different genesPol III: 30-50 genes

(Also- Organelle RNAPs in Mitochondria, Chloroplasts. Encoded by organelle genomes. Similar to bacterial RNAPs.)

Subunit structure of nuclear RNA polymerases

All three yeast polymerases have four coresubunits that exhibit some homology with the b,b‘, and a subunits in E. coli RNA polymerase.

The largest subunit (L’) of RNA polymerase II alsocontains an essential C-terminal domain (CTD). 26(yeast) to 52 (human) copies of (YSPTSPS).Phosphorylation site important for transcription.

RNA polymerases I and III contain the same twononidentical a -like subunits, whereas polymeraseII has two copies of a different a -like subunit.

All three polymerases share five other commonsubunits (two copies of the largest of these). Inaddition, each RNA polymerase contains four toseven unique smaller subunits.

Structure of eukaryotic RNA polymerase II

Taq RNAP

rRNA promoter transcribed by pol I

+1-150

UCE core45S pre-rRNA…

-150 UBF UBFSL1

Pol I

RNA pol III transcription:the 5S RNA gene

TFIIIB: contains TBP + 2 other subunits

- all three RNA pols use TBP!

TFIIIA: Zn-finger protein,Binds RNA and DNA

Primer extension assay

Mapping of Start Sites of MTE-containing Promoter

Mapping start sitesby in vitro

transcription

- + + +Initiation factors

Fractionation ofaccurate initiationactivity

Deletion analysis tolocate transcription-

control sequences

Plasmids with deletions of variouslengths 5’ to the transcription-start siteare then transfected into cultured cells(or used to prepare transgenicorganisms) and expression of thereporter gene is assayed.

The results of this hypotheticalexample (bottom) indicate that the testfragment contains two controlelements. The 5’ end of one liesbetween deletions 2 and 3; the 5’ endof the other lies between deletions 4and 5.

Yellow- 5’ promoterBlue- Reporter gene

Linker scanning mutations

Yellow- promoterBlue- Reporter geneOverlapping linkerscanning (LS) mutations(crosshatch) are introducedfrom one end of the regionbeing analyzed to theother. These mutationsresult from scrambling thenucleotide sequence in ashort stretch of the DNA.

Comparison of nucleotide sequences upstream of the start site in 60different vertebrate protein-coding genes.

Each sequence was aligned to maximize homology in the region from -35 to -20.The tabulated numbers are the percentage frequency of each base at each position.Maximum homology occurs over a six-base region, referred to as the TATA box,whose consensus sequence is shown at the bottom. The initial base in mRNAsencoded by genes containing a TATA box most frequently is an A. [See R.Breathnach and P. Chambon, 1981, Ann. Rev. Biochem. 50:349; P. Bucher, 1990,J.Mol. Biol. 212:563.]

Cis-acting control elements

(a) Genes of multicellular organisms contain both promoter-proximal elementsand enhancers as well as a TATA box or other promoter element.

(b) Most yeast genes contain only one regulatory region, called an upstreamactivating sequence (UAS), and a TATA box, which is ≈90 base pairs upstreamfrom the start site.

Core Promoter Elements including MTE

Enhancers and promoter-proximal elements in eukaryotic polII genes

Binding sites for activators that control transcription of the mouse transthyretin (TTR)promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. CellBiol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]

Transcriptional Regulation Requires Over 100 ProteinsThat Work in Concert to Trigger Proper Gene Activation

Major Points1. Three different RNA polymerases I,II and III direct synthesis of rRNA, mRNA and tRNA respectively2. Each RNA pol is a multisubunit protein complex that recognizes distinct promoter DNA sequences3. Primer extension is a sensitive, rapid and accurate assay to find the 5’ start site of transcription in vivo and in vitro4. Cell-free in vitro transcription assays (Run-off) provide a powerful tool to study the regulation of RNA synthesis5. Promoter mapping (linker scanning) and cell-based reporter gene assays help define composite Cis-regulatory elements6. Upstream enhancers, silencers, core promoters and downstream

elements make up complex, modular control DNA elements