1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 10
Eukaryotic RNA Polymerases and Their Promoters
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 10.1 Multiple Forms of Eukaryotic RNA Polymerase
There are at least two RNA polymerases operating in eukaryotic nuclei
One transcribes major ribosomal RNA 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 different compartment, the nucleolus

3. Separation of the 3 Nuclear Polymerases
Eukaryotic nuclei contain three RNA polymerases
These can be separated by ion-exchange chromatography
RNA polymerase I found in nucleolus
Location suggests it transcribes rRNA genes
RNA polymerases II and III are found in the nucleoplasm

4. Roles of the 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. RNA Polymerase Subunit Structures

6. Polymerase II Structure
For enzymes like eukaryotic RNA polymerases, can be difficult to tell:
Which polypeptides copurify with polymerase activity
Which are actually subunits of the enzyme
Epitope tagging is a technique to help determine whether a polypeptide copurifies or is a subunit

7. Epitope Tagging Add an extra domain to one subunit of RNA polymerase
Other subunits normal
Immunopreciptate with antibody directed against epitope
Denature with SDS detergent and separate via electrophoretic gel

8. Core Subunits of RNA Polymerase
Three polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activity (yeast)
Homologous to b’-, b-, and a-subunits (E.coli)
Both Rpb1 and b’-subunit binds DNA
Rpb2 and b-subunit are at or near the nucleotide-joining active site
Similarities between Rpb3 and a-subunit
There is one 20-amino acid subunit of great similarity
2 subunits are about same size, same stoichiometry
2 monomers per holoenzyme
All above factors suggest they are homologous

9. Common Subunits There are five common subunits
Rpb5
Rpb6
Rpb8
Rpb10
Rpb12
Little known about function
They are all found in all 3 polymerases which suggests they play roles fundamental to the transcription process

10. Summary
The genes encoding all 12 RNA polymerase II subunits in yeast have been sequenced and subjected to mutational analysis
Three of the subunits resemble the core subunits of bacterial RNA polymerases in both structure and function
Five are found in all three nuclear RNA polymerases, two are not required for activity and two fall into none of these categories

11. Heterogeneity of the Rpb1 Subunit
RPB1 gene product is subunit II
Subunit IIa is the primary product in yeast
Can be converted to IIb by proteolytic removal of the carboxyl-terminal domain (CTD) which is 7-peptide repeated over and over
Converts to IIo by phosphorylating 2 serine in the repeating heptad of the CTD
Enzyme with IIa binds to the promoter
Enzyme with IIo is involved in transcript elongation

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 DNA template
Catalytic center lies at the bottom of the cleft and contains a Mg2+ ion
A second Mg2+ ion is present in low concentration and enters the enzyme bound to each substrate nucleotide

13. 3-D Structure of RNA Polymerase II in an Elongation Complex
Structure of polymerase II bound to DNA template and RNA product in an elongation complex has been determined
When nucleic acids are present, the clamp region of the polymerase is 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

14. 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

15. Position of Critical Elements in the Transcription Bubble
Three loops of the transcription bubble are:
Lid: maintains DNA dissociation
Rudder: initiating DNA dissociation
Zipper: maintaining dissociation of template DNA

16. Proposed Translocation Mechanism
The active center of the enzyme lies at the end of pore 1
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

17. Structural Basis of Nucleotide Selection
Moving through the entry pore toward the active site of RNA polymerase II, incoming nucleotide first encounters the E (entry) site
E site is inverted relative to its position in the A site (active) where phosphodiester bonds form
E and A sites partially overlap
Two metal ions (Mg2+ or Mn2+) are present at the active site
One is permanently bound to the enzyme
The other enters the active site complexed to the incoming nucleotide

18. The Trigger Loop
In 2006 a crystal structure with GTP rather than UTP in the A site, opposite a C, revealed a part of Rpb1 roughly encompassing residues 1070 to a trigger loop
The trigger loop only comes into play when the correct substrate occupies the A site and makes several important contacts with the substrate that presumably stabilize the substrates association with the active site and contribute to the specificity of the enzyme

19. The Role of Rpb4 and Rpb7
Structure of the 12-subunit RNA polymerase II reveals that, with Rpb4/7 in place, the clamp is forced shut
Initiation occurs, with its clamp shut, it appears that the promoter DNA must melt to permit the template DNA strand to enter the active site
The Rpb4/7 extends the dock region of the polymerase, making it easier for certain general transcription factors to bind, thereby facilitating transcription initiation
Rpb7 can bind to nascent RNA and may direct it toward the CTD

20. 10.2 Promoters Three eukaryotic RNA polymerases have:
Different structures
Transcribe different classes of genes
We would expect that the three polymerases would recognize different promoters

21. Class II Promoters
Class II promoters are recognized by RNA polymerase II
Considered to have two parts:
Core promoter - attracts general transcription factors and RNA polymerase II at a basal level and sets the transcription start site and direction of transcription
Proximal promoter - helps attract general transcription factors and RNA polymerase and includes promoter elements upstream of the transcription start site

22. Core Promoter Elements – TATA Box
Very similar to the prokaryotic -10 box
Promoters have been found with no recognizable TATA box that tend to be found in two classes of genes:
1 - Housekeeping genes that are constitutively active in nearly all cells as they control common biochemical pathways
2 - Developmentally regulated genes

23. Core Promoter Elements
The core promoter is modular and can contain almost any combination of the following elements:
TATA box
TFIIB recognition element (BRE)
Initiator (Inr)
Downstream promoter element (DPE)
Downstream core element (DCE)
Motif ten element (MTE)
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

24. Elements
Promoter elements are usually found upstream of class II core promoters
They differ from core promoters in binding to relatively gene-specific transcription factors
Upstream promoter elements can be orientation-independent, yet are relatively position-dependent

25. 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

26. Class III Promoters
RNA polymerase III transcribes a variety of genes that encode small RNAs
The classical class III genes have promoters that lie wholly within the genes
The internal promoter of the type I class III gene is split into three regions: box A, a short intermediate element and box C
The internal promoters of the type II genes are split into two parts: box A and box B
The promoters of the nonclassical class III genes resemble those of class II genes

27. Promoters of Some Polymerase III Genes
Type I (5S rRNA) has 3 regions:
Box A, Short intermediate element, and Box C
Type II (tRNA) has 2 regions:
Box A and Box B
Type III (nonclassical) resemble those of type II

28. 10.3 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

29. Enhancers
Enhancers act through the proteins that are bound to them, enhancer-binding proteins or activators
These proteins appear to stimulate transcription by interacting with other proteins called general transcription factors at the promoter that promote the formation of a preinitiation complex
Enhancers are frequently found upstream of the promoter they control although this is not an absolute rule

30. Silencers
Silencers, like enhancers, are DNA elements that can act at a distance to modulate transcription but they inhibit, rather than stimulate, transcription
It is thought that they work by causing the chromatin to coil up into a condensed, inaccessible and inactive form thereby preventing the transcription of neighboring genes

31. Vital theme
The finding that a gene is much more active in one cell type than another leads to an extremely important point: All cells contain the same genes, but different cell types differ greatly from one another due to the proteins expressed in each cell
The types of proteins expressed in each cell type is determined by the genes that are active in those cells
Part of the story of the control of gene expression resides in the expression of different activators in different cell types that turn on different genes to produce different proteins