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Molecular BiologyFifth Edition
Chapter 10
Eukaryotic RNA Polymerases and Their Promoters
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
10-2
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
10-3
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
10-4
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
10-5
RNA Polymerase Subunit Structures
10-6
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
10-7
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
10-8
Core Subunits of RNA Polymerase
• Three polypeptides, Rpb1, Rpb2, Rpb3 are absolutely required for enzyme activity (yeast)
• Homologous to ’-, -, and -subunits (E.coli)• Both Rpb1 and ’-subunit binds DNA• Rpb2 and -subunit are at or near the
nucleotide-joining active site• Similarities between Rpb3 and -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
10-9
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
10-10
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
10-11
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
10-12
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
10-13
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
10-14
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
10-15
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
10-16
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
10-17
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
10-18
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 1100 - 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
10-19
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
10-20
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
10-21
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
10-22
Core Promoter Elements – TATA Box
• 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
10-23
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
10-24
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
10-25
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
10-26
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
10-27
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
10-28
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
10-29
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
10-30
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
10-31
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