RR3: gene regulatory sequences in RNA Pol II promoters Flashcards
what is a TATA sequence?
directs transcription at the promoters of some protein coding genes
not present in all genes, only in ones that are highly transcribed/heavily expressed
roughly at the same position: -35 to -25
what does it mean to be cis-acting or trans acting?
cis acting is within the DNA, contained within the gene
trans acting is when it comes from the exterior
what are the other proximal cis acting elements that drive transcriptional initiation?
the BRE TFIIB recognition element (-27 to -32)
the TATA box -31 to -26
the Inr initiator -2 to +4
the DPR (downstream promoter element) +28 to +32
how can transcriptional regulatory regions be identified?
the 5’ end can be cut shorter and shorter to create different segments of different lengths to try to identify where the regulatory sequences are
create a 5’ deletion series -> change the amount of regulatory sequence on the 5’ end
done with recombinant DNA techniques
ligate the different length promoters into a vector that carries a reporter gene (encodes an easily measured enzyme activity like beta gal)
transform E.coli and isolate plasmid DNAs
the promoters will drive the expression of the reporter gene downstream and give a read out of how effective transcription is
based on the expression of the gene, we can make conclusions on where the regulatory regions are
what is the limitation of that technique (5’ end deletion)
it changes the spacing/proximity
it is also quick and dirty, coarse analysis, not fine analysis
what are the different elements that can regulate eukaryotic genes?
exons, introns, TATA box, promoter proximal elements (up to -200b) enhancers (up to -50kb in mammalian genes) they are very far
in yeast, there is a regulatory element called UAS (upstream activating sequence) that works like an enhancer, but is closer than mammalian ones
also enhancers downstream, +10 to +50kb
what are enhancers?
cis acting elements that control tissue specific or stage specific transcription
some enhancers are conserved throughout vertebrates, the sequence is essential
how enhancers that are far away regulate transcription?
they are sometimes kilobases away from their targets
because chromosomes can form loops that results in regions that are usually linearly very distant to become physically adjacent to one other
the loops are associated with active transcription
enhancers can help to stabilise, generate and increase the rate of transcription within those loops, even if they are far from the target
what do mammalian genes that do not have a TATA box have?
they have regions upstream of the start sites that are rich in CpG (CGCGCGC)
CpG island promoters
the most common type of promoter in mammalian cells
not very efficient
where are CpG island promoters utilised?
for genes that do not have to be expressed at a very high copy number (housekeeping genes)
what is the “weird” thing about these CpG island promoters? and what might this explain?
they can send RNA polymerase in both directions, which is called divergent transcription
might explain why 80% of the genome is transcribed
learn how does linker scanning mutations work
(see slides)
