Eukaryotic txn I

In eukaryotes (as in pro) the major (?) point at which gene expression is controlled is at the level of transcription

-          Control is by DNA sequences and proteins that bind them

-          Usully more sequences (and thus more proteins binding)

-          DNA sequences can be very far away from promoter it is regulating (kb away)

Eukaryotic RNAPs

-          eukaryotes have 3 RNAPs

o       found by fractionating nuclear extracts by column chromatography

o       RNAP I, II, III

§         RNAP I

·         Makes pre-rRNA (processed to 28S, 5.8S and 18S rRNA)

·         rRNA genes are found as tandem repeats and the DNA is found in a distinct region of the nucleus, the nucleolus

·         each has own promoter upstream of txn start site

§         RNAP III

·         Makes small RNAs like tRNAs and a few others

·         Txn control elements of these are inside the txn unit

§         RNAP II

·         Makes mRNAs that are translated

Cis vs trans acting elements review

DNA elements that control transcription

“Wreck and check” experiments

-          example

o       have putative txn control element on plasmid, driving expression of a reporter gene

o       make mutations in putative promoter region

§         5’ deletions

§         Can make site-directed mutations

o       Then ask, how active is promoter?

§          Assay by reporter gene product such as b-galactosidase, luciferase, or green fluorescent protein

DNA elements

TATA box

-          find TATA boxes when look for consensus sequences, around 30-50bp upstream of start site

-          TATAA/TAA/TA/G

-          Often find several transcription start sites for genes

Initiator

-          sequence right around start site

-          not found in promoters with TATA boxes

-          very degenerate consensus sequence

Housekeeping genes

-          many genes transcribed at a constant low rate, usually for essential enzymes needed all the time at constant levels

-          often don’t have TATA or initiator elements and txn start sites spread out over long distance

THIS BOOK CALLS TATA/INITIATOR AND TXN START SITE THE PROMOTER

Promoter-proximal elements

-          within 100-200 bp of start site

-          can monkey around with spacing between these and TATA box much more than CAP-TATA in lac operon, although not unlimited

Enhancers

-          SV40 a approx. 100bp sequence was found about 100bp upstream of transcription start site

-          Found that this DNA element can increase transcription from the SV40 promoter when tens of thousands of bp away and in either orientation (even downstream), as long as on same piece of DNA

-          Then found that it can stimulate txn from a wide variety of promoters that have proximal elements

-          Called enhancer

o       mutagenesis says that many sequences within the enhancer are important and that each contributes a bit of activity (protein binding sites)