1. Eukaryotic RNA Polymerases and their Promoters
Chapter 10

2. Multiple Forms of Eukaryotic RNA Polymerase – Early studies
There are at least two RNA polymerases operating in eukaryotic nuclei
One transcribes major ribosomal RNA (rRNA) genes
One or more to transcribe rest of nuclear genes
Ribosomal genes are different from other nuclear genes
Different base composition from other nuclear genes
Unusually repetitive
Found in the nucleolus
Depending on the organism each cell contains from several hundred to over 20,000 copies of rRNA gene. They are found in nucleolus and rest of the nuclear genes are present in nucleoplasm

3. Separation of the Three Nuclear Polymerases
Eukaryotic nuclei contain three RNA polymerases
Separated by ion-exchange chromatography
RNA polymerase I found in nucleolus
transcribes rRNA genes
RNA polymerases II and III are found in the nucleoplasm
- transcribes other kinds of RNA
Robert Roeder and William Rutter in 1969 performed DEAE-Sephadex ion-exchange chromatography and studied three peaks of polymerase activity in order of their emergence from ion-exchange column.

4. Roles of three RNA Polymerases
Polymerase I makes large rRNA precursor
Polymerase II makes
Heterogeneous nuclear RNA (hnRNA)
Small nuclear RNA
Polymerase III makes precursors to tRNAs, 5S rRNA and other small RNA

5. Polymerase Structure? Hard to tell:
Which polypeptides copurify with polymerase activity?
Which are actually subunits of the enzyme?
Technique to help determine whether a polypeptide copurifies or is a subunit is called epitope tagging
For enzymes like eukaryotic RNA polymerases, can be difficult to tell

6. RNA Polymerase Subunit Structures
Pierre Chambon and Rutter studied RNA polymerase II in great detail. In prokaryotes it was possible to separate the subunits and study what is required to reconstitute an eukaryotic nuclear polymerase from its separate subunits. This could not be performed in eukaryotic RNA polymerase. The other approach tried was mutating the subunits and determining what is required for what activity? Altogether 12 subunits have been found in yeasts and humans

7. Epitope tagging-Richard Young
Add an extra domain to one subunit
Other subunits normal
Polymerase labeled by growing in labeled amino acids
Purify with antibody
Denature with detergent and separate on a gel

8. Polymerase II Young - 10 subunits are placed in 3 groups:
Core – (3 of the subunits) - related in structure and function to bacterial core subunits
Common – (5 of the subunits) - found in all 3 nuclear RNA polymerases in yeast
Nonessential subunits – (2 of the subunits) - conditionally dispensable for enzymatic activity

9. Core Subunits
Three polypeptides - Rpb1, Rpb2, Rpb3 -absolutely required for enzyme activity
These are homologous to b’-, b-, and a-subunits
Both Rpb1 and b’-subunit binds DNA
Rpb2 and b-subunit are at or near the nucleotide-joining active site
Rpb3 does not resemble a-subunit
There is one 20-amino acid subunit of great similarity
2 subunits are about same size - same stoichiometry

10. Common Subunits There are five common subunits
Rpb5
Rpb6
Rpb8
Rpb10
Rpb12
Little known about function
They are all found in all 3 polymerases
Suggests play roles fundamental in transcription
They are all found in 3 polymerases in yeasts

11. Subunits Nonessential for Elongation
Rpb4 and Rpb7
Dissociate fairly easily from polymerase
Might shuttle from one polymerase II to another
Rpb4 may help anchor Rpb7 to the enzyme
Mutants without Rpb4 and Rpb7 transcribes well- but cannot initiate at a real promoter
Rpb7 is an essential subunit
Rpb7 is an essential subunit - so must not be completely absent in the mutant

12. The Three-Dimensional Structure of RNA Polymerase II
Structure of yeast polymerase II (pol II 4/7) - reveals a deep cleft that accepts a linear DNA template from one end to another
Catalytic center lies at the bottom of the cleft and contains a Mg2+ ion
Upper jaw – Rpb1+Rpb9 and lower jaw – Rpb5
Geometry allows enough space for:
TFIID to bind at the TATA box of the promoter
TFIIB to link the polymerase to TFIID
Places polymerase correctly to initiate transcription
It contains two Mg 2+ ions but one is lesser in concentration. Cleft has basic residues which bind the enzyme to acidic DNA template. When nucleic acids are present, the clamp region of the polymerase has shifted closed over the DNA and RNA
Closed clamp ensures that transcription is processive – able to transcribe a whole gene without falling off and terminating prematurely.

13. Position of Nucleic Acids in the Transcription Bubble
DNA template strand is shown in blue
DNA nontemplate strand shown in green
RNA is shown in red

14. Position of Critical Elements in the Transcription Bubble
Three loops of the transcription bubble are:
- Rudder: initiating RNA- DNA dissociation
Lid: maintains RNA-DNA dissociation
Zipper: maintaining dissociation of template DNA
Formation and maintenance of transcription bubble and dissociation of RNA-DNA hybrid. If the RNA-DNA hybrid extended farther than 9 bp, the rudder would be in the way. Rudder facilitate dissociation of hybrid.

15. Transcription mechanism
Pore 1 also appears to be the conduit for:
Nucleotides to enter the enzyme
RNA to exit the enzyme during backtracking
Bridge helix lies next to the active center
Flexing this helix may function in translocation during transcription
The active center of the enzyme lies at the end of pore 1. A polymerase can pause and then move backwards extruding the 3’ end of nascent RNA out of the enzyme.There is a pore on bottom of enzyme called pore 1 that might serve as exit point for RNA extrusion. Backtracks happen when nucleotide is misincorporated, thus exposing misincorporated nucleotide to removal by TFIIS which binds to the funnel at the other end of pore 1.

16. Class II promoters
Class II Promoters - recognized by RNA polymerase II - are similar to prokaryotic promoters
Considered to have two parts:
Core promoter having 4 elements
Upstream promoter element

17. Core Promoter Elements – TATA Box
Found on the nontemplate strand
Very similar to the prokaryotic -10 box
There are frequently TATA-less promoters
Housekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathways
Developmentally regulated genes

18. Other core elements
- TFIIB recognition element (BRE)
- Initiator (Inr)
- Downstream promoter element (DPE)
- At least one of the four core elements is missing in most promoters
- TATA-less promoters tend to have DPEs
- Promoters for highly specialized genes tend to have TATA boxes
Promoters for housekeeping genes tend to lack them

19. Upstream promoter
Upstream promoter elements are usually found upstream of class II core promoters
Differ from core promoters in binding to relatively gene-specific transcription factors
GC boxes bind transcription factor Sp1
CCAAT boxes bind CTF (CCAAT-binding transcription factor)

20. Class I promoters
Class I promoters are not well conserved in sequence across species
General architecture of the promoter is well conserved – two elements:
Core element surrounding transcription start site
Upstream promoter element (UPE) 100 bp farther upstream
Spacing between these elements is important

21. Three types of class III promoters
Type I (5S rRNA) has 3 regions:
Box A
Short intermediate element
Box C
Type II (tRNA) has 2 regions:
Box B
Type III (nonclassical) resemble those of type II
RNA polymerase III transcribes a set of short genes
These have promoters that lie wholly within the genes
There are 3 types of these promoters

22. Enhancers and Silencers
These are position- and orientation-independent DNA elements that stimulate or depress, respectively - transcription of associated genes
Are often tissue-specific in that they rely on tissue-specific DNA-binding proteins for their activities
Some DNA elements can act either as enhancer or silencer depending on what is bound to it

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