regulatory sequences in RNA pol II promoters

cis-acting regulatory elements

• BRE (TFIIB recognition element)

• TATA box

• Inr (initiator)

• DPE (downstream promoter element)

mammalian gene with TATA box

highly transcribed

mammalian gene with CpG island promoter

housekeeping gene

upstream activating sequence (UAS)

regulatory element in many yeast genes that works like an enhancer

transformation

introduction of recombinant plasmid vector into bacteria

transfection

introduction of recombinant plasmid vector into mammalian cells

transgenics

introduction of recombinant plasmid vector into live animals/plants

transthyretin

important transport protein that carries vitamin A and thyroxine

identification of transcriptional regulatory regions

using recombinant DNA techniques, a series of DNA segments corresponding to regions upstream of the transcription start site are used to identify important control elements, where each segment is transfected into a cell and monitored for reporter enzymatic activity

reporter gene

encodes an easily measured enzyme activity

linker scanning mutations to identify cis-acting regulatory sites

different regions of the control region are mutated (preventing proper expression of those regions) and level of reporter gene transcription is observed

common reporters

• green fluorescent protein (GFP)

• β-galactosidase (lacZ)

• thymidine kinase (tk)

• luciferase (luc)

• chloramphenicol acetyltransferase (CAT)

enhancers

cis-acting elements that control tissue-specific or stage-specific transcription

function of chromatin loops

• regions that are linearly very distant can be physically adjacent to one another in 3D space

• often associated with active transcription

• enhancers may help to generate, stabilize, and increase rate of transcription within loops, while the genes they affect may be linearly distant