Transcription control elements (DNA sequences) are binding sites for transcription factors, proteins that regulate transcription from an associated promoter.

Activators stimulate transcription.

Repressors inhibit transcription.

AATTGGGGCGGGGC CCCCGCCCCGTTAA( )n

Hybridize andligate

AATTGGGGCGGGGCAATTGGGGCGGGGCAATTGGGGCGGGGC…………………… CCCCGCCCCGTTAACCCCGCCCCGTTAACCCCGCCCCG……………………

AATTGGGGCGGGGCAATTGGGGCGGGGCAATTGGGGCGGGGC…………………… CCCCGCCCCGTTAACCCCGCCCCGTTAACCCCGCCCCG……………………

Specific sequence DNA affinity column

Covalently link synthetic,Ligated DNA to a solid support

Obtain amino acid sequence from isolated transcription factor.

Search the cDNA (EST) data base for a cDNA that encodes the transcription factor.

Insert this into an expression vector.

Assay the ability of the transfected expression vector to stimulate reporter gene expression from a co-transfected reporter plasmid.

WT or mutant Gal4

Gal4s

WT or mutant Gal4

Expression vector:

Fig. 11-17

Fig. 11-17

E. coliLexA

yeast Gal4aa738--823

CN

E. coliLexA

CN

Expression vector for:

or

LexA bindingsites

:

Gal4 reporterLexA reporter

LexA vector

g

alac

tosi

das

e u

nit

s

400

800

1200

LexA-Gal4(738-823) vector

+___

+ +

++

_

___

_

_+

+

1600

2000

Fig. 11-18

Fig. 4-17

Fig. 11-20

Interface of 434 repressorwith operator DNA(lower monomerand dimer Interface)

Fig. 3-6

Fig. 2-13

Sequences of C2H2 zinc-fingers in TFIIIA

Fig. 11-21

Zinc-finger protein(C2H2 type)