1. Conclusions (last lecture)
Conserved structure of RNA pol II
RNA pol II must navigate a chromatin template in a highly dynamic environment

2. Outline Mechanism of Transcription Activation
RNA pol II structure and function
Coordination of RNA processing
Transcription Regulatory elements
Identification & characterization in vivo
Examples of mammalian transcriptional control regions
Transcription Factors

3. Central Dogma Why aren’t tRNAs and rRNAs processed like mRNAs?
What is different about how mRNAs are made?
Fig. 1-9 Lodish 2013

4. Only RNA Polymerase II has a C-Terminal Domain
CTD is Phosphorylated Prior to Transcription Initiation
CTD
Red arrow indicates location where unique C-terminal
domain extends from in RNA pol II
Fig Lodish 2013

5. The RNA Pol II C-Terminal Domain (CTD) facilitates capping, splicing and polyadenylation by recruitment of RNA processing factors.
Hsin J , and Manley J L Genes Dev. 2012;26:
©2012 by Cold Spring Harbor Laboratory Press

6. 5’ Capping Splicing 3’ Processing
RNA Pol II CTD phosphorylation coordinates the transcription cycle with pre-mRNA processing and histone modification.
5’ Capping
Splicing
3’ Processing
Hirose Y , Ohkuma Y J Biochem 2007;141:
© 2007 The Japanese Biochemical Society.

7. Drosophila chromosome stained with antibody to phosphorylated Pol II (Red) or unphosphorylated Pol II (Green)
What do you notice?
Fig Lodish 2013

8. Central Dogma
Fig. 1-9 Lodish 2013

9. Alternative Splicing Simple Complex
Which promoter is regulating mRNA2 transcription?

10. Pax6: Master regulator of eye development
Ectopic Expression of Eyeless (PAX 6 homolog)
in Drosophila results in aberrant formation of eye structures
Antenna Lower leg
Halder et al. Science 267: 1788 (1995)

11. Different Pax 6 enhancers are active in different tissues:
Results in different sites of transcription initiation and
alternative splicing in different tissues
Transcript a is detected in lense
and pancreas. Lacks exons 1 and α
PAX6: paired box and Homeobox DNA binding domain – highly conserved in mammals, flies and worms
Mammals: required for retina, brain and pancreatic beta cells (Pax 6 +/-) = lack of irisLP = lens pit.
Actually there are 3 different promoters utilized at different times during embryogenesis in diff tissues of the developing embryo
Transcript in Lens and pancreas initiates at exon 0
Transcript in brain initiates at exon 1
Transcript in retina initiates at exon alpha (unique to vertebrates so invertebrates cannot restrict light by constricting retina in response to excessive light, must close eye) – may have been an evolutionary advantage in vertebrates.
Aniridia (loss of iris) occurs in 1/60,000 individuals
Fig. 7-6 Lodish et al. 2008

12. How can we study gene expression in vivo?
One method: transgenic mouse
Introduce “reporter gene” controlled by potential regulatory elements into a mouse
transgenic mouse animation
Distinct from reporter gene assay which is performed in cell culture

13. Answer Pax6 gene questions in your course manual
What parts of the Pax6 gene contain regulatory elements?
Upstream region and introns
What DNA constructs could have been used to create the transgenic mice shown in the bottom panels of Fig. 7-6?
For the far left picture which shows staining in lens pit and pancreas: Could have used 8kb upstream of exon 0, directing expression of the beta gal gene (don’t need any other pax 6 sequences.
For the embryo showing staining in the retina, could have used the intron between exons 4 and alpha (intron 5), directing expression of the beta gal gene (don’t need any other pax6 sequences.
Take home point: these sequences are binding regulatory factors that are found only in certain tissues, and these are stimulating transcription in those tissues.
Could this same information have been obtained from an in vitro cell culture experiment? Why or why not?
Could get some of this info from different cell culture lines (like intestinal cells used in discussion section experiments) – but difficult to recreate cross-talk between tissues in vitro
What experiments could you perform to identify important transcription control regions? How would you know where to start looking?
Could take a comparative genomics approach and look at sequences that are highly conserved between mouse, human etc. or could look at individuals with mutant phenotypes (eyeless) and see where the mutations lie. Then could mutate these regions and look at effect on expression.

14. Alternative Pax 6 promoters & enhancers are used to generate
different transcripts in different tissues
Transcript c:
detected in retina
lacks exons 0-4
contains exon α
LP = lens pit.
Actually there are 3 different promoters utilized at different times during embryogenesis in diff tissues of the developing embryo
Transcript in Lens and pancreas initiates at exon 0
Transcript in brain initiates at exon 1
Transcript in retina initiates at exon alpha (unique to vertebrates (invertebrates cannot restrict light by constricting retina in response to excessive light, must close eye) – may have been an evolutionary advantage in vertebrates. (not present in worms, mollusks, insects)
Fig. 7-6 Lodish et al. 2008

15. Conclusions
Unique Pol II CTD provides a mechanism for coupling mRNA-specific processing with transcription
Different enhancers/promoters can control transcription of the same gene in different cell types
Different subsets of transcription factors bind to enhancers of the same gene in different cell types
Enhancers can be located far from transcription start sites