coding strand will be exposed so RNA pol II can start to read off its template and start making mRNA
Switch from pre-initiation to initiation
The protein kinase activity associated with TFIIH will phosphorylate key residues on the carboxy terminal domain
of RNA polymerase II and this phosphorylation is also associated with the switch from initiation to elongation
Helicase opens up a bubble, protein kinase is responsible for the phosphorylation
Components of the basal transcription machinery play additional roles
TFIIH is the only general/basal transcription factor that has ATP-dependent enzymatic activities
TFIIH contains two DNA helicases involved in Xeroderma pigmentosum
Xeroderma pigmentosum patients have a faulty nucleotide excision repair (NER) system
Some patients have defects that are much more severe
TFIIH may couple basal transcription with DNA repair
Heavily transcribed regions are repaired more
effectively
TFIIH may affect transcription-coupled repair
o
Would repair thymine dimer
Developmental gene expression is synchronized through release of paused elongation complexes
Transcription-initiation is probably the rate-limiting step, but it may not be a general case
Gene transcription may be regulated downstream of initiation at elongation
Instead of immediately going through the gene, the complex may get to the first nucleosome and wait for particular
contingencies to switch over to an elongation phase of active transcription
A number of genes tend to be transcribed, elongated at the same time
There is a coordination of gene expression associated with elongation controls as opposed to initiation controls
What genes are being actively transcribed?
Nuclear Run on or Genome-wide Nuclear Run on (GRO)
You purify nuclei and provide those nuclei with a radioactively-labelled nucleotide which will be incorporated into a
growing mRNA chain of RNA pol complexes that are actively transcribing any gene at any given time
You let that go for a while then you stop it and add elongation inhibitors
You can purify RNA from that reaction and get an idea of how many RNA pol molecules were transcribing
–
should
be more or less related to the amount of radioactive RNA that you get from that particular sample
GRO can be combined with RNAseq
–
a genome-wide run-on assay to figure out how well all the genes are being
transcribed at one single time
Instead of radiolabelling all the RNAs at once, you can use antibodies
o
Antibodies against Phospho-RNA Polymerase II (elongation specific)
Antibodies recognize elongating RNA Pol II
Smash up nuclei and add antibodies so they can interact with its antigen RNA Pol II in its elongating form
You can get rid of everything else and pull down RNA pol II and presumably all the RNA still associated with
it
o
Perform immunoprecipitation and recover RNA
Get rid of everything else
Can bind antibody to certain matrices
Spin complexes and precipitate them
