Regulatory sequences in RNA Pol II (3)

Polythene chromosomes

Multiple copies of DNA strands that are held together

Banding pattern in Drosophila

Puffs = active transcription

Stained polythene chromosomes with antibodies that recognizes phosphorylated CTD (red)

Antibody only binds to puffs

Lac operon

Gene that is turned on when glucose is absent but lactose is present

Specificity of RNA Pol in lacZ

Given by sigma factors

Position of TATA box

35-25 bases upstream of start site

How was the importance of the TATA box proved?

Genes missing the TATA box were not expressed efficiently

Other cis-acting promoters

BRE (-37 to -32), Inr (-2 to 4) and DPE (28 to 32)

Why cis-acting elements?

Regulate the expression of the gene that is downstream from them

Position of the enhancer sequences

- or + 50 kb

10-15% of genes in organisms

Mammalian gene with TATA box and enhancers

50-75% of genes in organism

Mammalian gene with CpG islands

Specialty of CpG islands

Drive transcription both ways

Yeast UAS

Enhancer

Transformation

DNA transfer in bacteria (prokaryotic)

Reporter gene use

Indicates when transcription is active

Reporter gene protein level (translation) is proportional to…

Its activation

Transfection

DNA introduction in mammalians (eukaryotic)

Transgenic

DNA introduction into live animals/plants

TTR

Gene

5’-deletion series

Deleting upstream region from the 5’ end (PCR) to identify important elements

Result of reporter-gene expression

1 important element is 2.2-3 kb and another at 0.7-1.5 kb

Linker scanning mutation

More accurate than 5’-deletion series and preserves spatial relationships between regions (helix)

Common reporter genes (track expression of specific gene)

GFP and lacZ

Distance (genes in the 50kb) and enhancers

Activation still happens (important in tissue specific transcription)

Enhancers also influence…

Stage-specific transcription (limbs)

How can enhancers still work at a distance?

Genes are NOT linear: loops allow genes and corresponding enhancers to be adjacent