Molecular Biology Fifth Edition Chapter 10 Eukaryotic RNA Polymerases and Their Promoters Lecture PowerPoint to accompany Robert F. Weaver Copyright ©

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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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