1. Some Nov.16 study questions:
Do you know the basic structure of eukaryotic gene regulatory regions? Do you know the differences from prokaryotes?
Do you know the main components of the eukaryotic Pol II transcription complex and their functions?
Do you understand the job of mediator?
Do you understand the Pol II CTD phosphorylation cycle and the kinases involved?

2. “What is true in E. coli must also be true in elephants.”

3. “What is true in E. coli must also be true in elephants.”
Eukaryotic transcription conserves basic principles from bacteria
“What is true in E. coli must also be true in elephants.”
Jacques Monod, Nobel Prize 1965

4. Department of Pathology
Lecture - Nov. 16, 2016
Dean Tantin, PhD
Department of Pathology
University of Utah
JMRB 5700B
Transcriptional Regulation in Eukaryotes Concepts, Strategies, and Techniques, Second Edition

5. Yeast Pol II transcribing into a nucleosomal array
Transcriptional Regulation in Eukaryotes Concepts, Strategies, and Techniques, Second Edition

6. For exam, things you are NOT responsible for:
You do not need to know the order of assembly of the different GTFs and polymerase on DNA (it is not known how much this is dependent on the in vitro system and how it assembles in vivo). I will use ordered assembly to gradually build up the complex so you can see different structures and functions.
For GTFs composed of multiple subunits, you do not need to know their individual functions (you do need to know that TFIID=TBP+TAFs and the jobs for each GTF).
You do not need to know the individual TAF subunits (just what TAFs do generally).
You do not need to know the individual mediator subunits (just what the different mediator modules do generally).
You are not responsible for transcription doohickeys not mentioned/shown in the lecture (TFIIA, Spt4/5, NELF, DSIF, PAF, SEC, PC1, NC1, etc.)

7. Bacterial transcription: s70 positions the polymerase core on the DNA
A classic bacterial
s70 promoter includes -10 and -35 control regions
Sigma region 4 binds -35
Sigma region 2 binds -10
Eukaryotic transcription is much more complex…

8. France: a b c Robert Roeder Pierre Chambon
Column chromatography separates and identifies the three eukaryotic RNA polymerases, each with its own sensitivity to a-amanitin
Robert Roeder
Pierre Chambon

9. Robert Roeder Bill Rutter lab
Sephadex A25 chromatography of the three nuclear RNA polymerases from sea urchin embryos. Original graph from 18 February, Red, RNA polymerase activity; blue, total protein; black, ammonium sulfate gradient.
Robert Roeder
Bill Rutter lab

10. RNA polymerase I 5.8S, 18S, 28S rRNA
RNA polymerase II all protein-coding genes, snoRNA, some snRNA,
pre-microRNAs, lncRNAs…
RNA polymerase III tRNA, 5S rRNA, some snRNA, genes for other
small RNAs

11. Organization of eukaryotic protein-coding genes
A classic bacterial
s70 promoter includes -10 and -35 control regions
General organization of control elements that regulate gene expression in multicellular eukaryotes and yeast.
In eukaryotes, the term “promoter” refers to all TSS-proximal regulatory elements, positive and negative (e.g., analogs of the CAP site, promoter and operator). approx to +50. “Core promoter”: approx. -50 to +10

12. TATA box is recognized by TATA box binding protein, TBP.
1980s: the search for eukaryotic analogs of s factors, and bacterial regulatory elements such as the -10/-35 sequence, helped identify components of the transcription complex, including TBP.
A weight matrix:
TATA box is recognized by TATA box binding protein, TBP.
Not all promoters have TATA boxes, but TBP is required in all cases.
The TATA box: Pierre Chambon
1980 Proc. Natl Acad. Sci. USA 77:7024

13. TATA box is recognized by TATA box binding protein, TBP.
Dark blue/red:
Phosphate backbones
Light blue/red:
Corresponding bases
Dashed line:
Pseudodyad axis
TATA box is recognized by TATA box binding protein, TBP.
Not all promoters have TATA boxes, but TBP is required in all cases.
TBP: 1989 Horikoshi et al., Cell 341:299
(Structure by Steve Burley 1993)

14. TATA box is recognized by TATA box binding protein, TBP.
Distortion in DNA caused by phenylalanines introduced into minor groove
TATA box is recognized by TATA box binding protein, TBP.
Not all promoters have TATA boxes, but TBP is required in all cases.
TBP: 1989 Horikoshi et al., Cell 341:299
(Structure by Steve Burley 1993)

15. Space filling view, with artificial B-DNA extensions
TATA box is recognized by TATA box binding protein, TBP.
Not all promoters have TATA boxes, but TBP is required in all cases.
Later TBP was shown to be required by all three polymerases.

17. TBP—green (TAFs not shown); TFIIB—yellow
Carey (2012) Nat. Struct. Mol. Biol. 19:737

18. TBP+TAFs=TFIID ? TBP—green (TAFs not shown); TFIIB—yellow
Carey (2012) Nat. Struct. Mol. Biol. 19:737

19. For
Your
Reference
Transcriptional Regulation in Eukaryotes Concepts, Strategies, and Techniques (2nd Ed.)

20. TAFs…what you need to know
TAFs talk to chromatin.
Example: Tandem bromodomains of TAF1
Example: PHD finger of TAF3 recognizes H3K4me3
TAFs can talk to sequence-specific transcription factors
Canonical TAFs are conserved from humans to yeast ?
But…
TAFs are NOT necessary for most transcription (unlike TBP).
TAFs need NOT be TBP-associated.
TAFs can operate away from promoters.
Different cell types express different sets of canonical and non-canonical TAFs.
TAFs remain something of a black box.

22. Red:TBP, Yellow:TFIIB Core, Green:TFIIB Ribbon, Gray: Pol II, Pink:DNA
TFIIB and TBP position promoter DNA adjacent to Pol II cleft
TFIIB is important for positioning the transcription initiation site.
modeled B-DNA
transcription
Red:TBP, Yellow:TFIIB Core, Green:TFIIB Ribbon, Gray: Pol II, Pink:DNA

23. Organization of eukaryotic protein-coding genes

24. Only the two largest (of 12) subunits of Pol II are shown
Only the two largest (of 12) subunits of Pol II are shown. TFIIF (a two-subunit elongation factor) is also shown.
Rpb1 and Rpb2 together form the active site into which the template strand will be inserted
TBP—green (TAFs not shown); TFIIB—yellow; Pol II—gray, the two largest subunits only are shown for simplicity; active site--orange; TFIIF—olive
Carey (2012) Nat. Struct. Mol. Biol. 19:737

25. CTD of largest subunit extends from position of red arrow.
Comparison of three-dimensional structures of bacterial and eukaryotic RNA polymerases
10/12 subunits.
CTD of largest subunit extends from position
of red arrow.
12/12 subunits.
“Clamp” covers template DNA so polymerase does not release.
“Wall” forces coding strand DNA to bend.

26. NO!
Schematic representation of the subunit structure of the E. coli RNA core polymerase and yeast nuclear RNA polymerases

27. The CTD: large and involved in multiple co-transcriptional events
(YS2PTS5PS)52
Major sites of regulation during the transcription cycle.
Phatnani & Greenleaf, 2006
Prolines prevent compaction into helices and sheets
Proline isomerization can alter conformation
Transcription in eukaryotes is tightly coupled to
RNA processing (capping, splicing, polyadenylation)
in part through the CTD

28. The CTD: somebody way-back-when thought more was better
(YS2PTS5PS)52
RA Young, Ann. Rev. Biochem. 1991

29. (YS2PTS5PS)52 major transcription regions
Antibody staining demonstrates that the carboxyl-terminal domain (CTD) of RNA polymerase II is phosphorylated during in vivo transcription
un-phos Ab—green, ser2-phos Ab—red
(YS2PTS5PS)52

30. TBP—green (TAFs not shown); TFIIB—yellow; Pol II—gray, the two largest subunits only are shown for simplicity; active site of Rpb1--orange; TFIIF—olive; TFIIE—red; TFIIH (one subunit)—dark orange.
Carey (2012) Nat. Struct. Mol. Biol. 19:737

31. TFIIH subunit structure: -9-10 subunits
-Three broad domains, one with kinase activity (top) and one with two helicase activities (middle), and one that attaches to DNA and the rest of the transcription comlex (bottom).
Gibbons et al. (2012) PNAS 109:1949

32. All you need to know about these knobs and cylinders: TBP binding the TATA box. TFIIB positions Pol II. TFIIF acts as an elongation factor. TFIIE and part of TFIIH grab hold of the coding strand at the bubble region and stabilize it. The TFIIH helicases use the energy of ATP hydrolysis to pump DNA into the active site. Simultaneously, the kinase portion of TFIIH phosphorylates the Pol II CTD on Ser5.
Carey (2012) Nat. Struct. Mol. Biol. 19:737

33. a-b hydrolysis for RNA synthesis

34. a-b hydrolysis for RNA synthesis
b-g hydrolysis for kinase/helicase activity
Pol II CTD phosphor-ylation on Ser5 by e.g. Cdk7 (TFIIH) or Cdk8 (Mediator)

35. -- Closed complex (D/B/Pol/F/E/H) formation
ATPADP+Pi open complex formation (TFIIH)
ATPADP+P trans Ser5 CTD phosphoryation (TFIIH and/or mediator)
NTPNMP+PPi Abortive initiation
ATPADP+P trans Ser2 CTD phosphorylation (pTEFb=CDK9+cyclinT)
NTPNMP+PPi Promoter clearance (AKA promoter escape)
These steps are thought to happen at the same time
These steps are thought to be regulated

36. 7SK is an abundant small nuclear noncoding RNA.
It forms a small nuclear ribonucleoprotein complex (snRNP) with other proteins that regulate transcription by controlling pTEFb.

37. Contacts activation domains Pol II CTD Ser5 kinase
Cartoon-ish version of several DNA-bound activators interacting with the transcription complex
>30 subunits
Contacts polymerase
Contacts activation domains
Pol II CTD Ser5 kinase

38. Yeast mediator plus Pol II plus DNA:
Mediator middle region contains CTD kinase activity.
Pol II CTD contacts mediator head region.
Activators can contact mediator tail region.
Mediator: Roger Kornberg
1990 Cell 61:1205

39. (TFIIH also has a Ser5 kinase)
Mediator is more universally required for Pol II gene transcription than are TAFs
Pol II CTD
Ser5 kinase
(TFIIH also has a Ser5 kinase)
Activators like Gal4, Gcn4 and SREBP recruit mediator through Med15 (AKA Gal11) of the tail.
Contacts Pol II
Mediator: Roger Kornberg
1990 Cell 61:1205

40. Cartoon-ish version of several DNA-bound activators interacting with the transcription complex.