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.

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

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

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

RNA Polymerase Subunit Structures

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

10.3 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

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

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

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