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Molecular Biology Lecture 16 Chapter 10 Eukaryotic RNA Polymerases and Their Promoters Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Separation of the Three Nuclear Polymerases • Eukaryotic nuclei contain three RNA polymerases These can be separated by ion-exchange chromatography 10-2 Separation of the Three Nuclear Polymerases nucleoplasm nucleolus •RNA polymerase I found in nucleolus –Location suggests in transcribes rRNA genes 10-3 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 10-4 RNA Polymerase Subunit Structures 10-5 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 • Technique to help determine whether a polypeptide copurifies or is a subunit is called epitope tagging 10-6 Epitope Tagging • 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 10-7 10-8 Polymerase II Original 10 subunits are placed in 3 groups: • Core – related in structure and function to bacterial core subunits • Common – found in all 3 nuclear RNA polymerases • Nonessential subunits – conditionally dispensable for enzymatic activity 10-9 Core Subunits • Three polypeptides, Rpb1, Rpb2, Rpb3 are 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 – 2 monomers per holoenzyme 10-10 Common Subunits • There are five common subunits – Rpb5 – Rpb6 – Rpb8 – Rpb10 – Rpb12 • Little known about function • They are found in all 3 polymerases • Suggests play roles fundamental in transcription 10-11 Subunits Nonessential for Elongation • Rpb4 and Rpb7 – – – – – Dissociate fairly easily from polymerase Found in substoichiometric quantities 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, so must not be completely absent in the mutant 10-12 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 carboxylterminal domain (CTD) which is 7aa-peptide repeated over and over. Enzyme with IIa binds to the promoter – Converts to IIo by phosphorylating 2 ser in the repeating heptad of the CTD. Enzyme with IIo is involved in transcript elongation 10-13 The Three-Dimensional Structure of RNA Polymerase II • Structure of yeast polymerase II (specifically pol II 4/7) at atomic resolution reveals a deep cleft that accepts a linear DNA template from one end to another 10-14 • Catalytic center lies at the bottom of the cleft and contains a Mg2+ ion • A second Mg2+ ion present in low concentrations • 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 10-15 Promoters • Three eukaryotic RNA polymerases have: – Different structures – Transcribe different classes of genes • Expect that the 3 polymerases would recognize different promoters 10-16 Class II Promoters • Promoters recognized by RNA polymerase II (class II promoters) are similar to prokaryotic promoters • Considered to have two parts: – Core promoter having 4 elements – Upstream promoter element 10-17 10-18 Core Promoter Elements – TATA Box • 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 10-19 Core Promoter Elements • In addition to TATA box, core promoters are: – 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 10-20 Upstream Elements • 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) • Upstream promoter elements can be orientationindependent, yet are relatively positiondependent 10-21 Upstream Elements 10-22 Enhancers and Silencers • These are position- and orientationindependent 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 10-23 10-24 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 RNA polymerase 1 promoters 10-27 10-28 Class III Promoters • 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 10-29 Promoters of Some Polymerase III Genes • Type I (5S rRNA) has 3 regions: – Box A – Short intermediate element – Box C • Type II (tRNA) has 2 regions: – Box A – Box B • Type III (nonclassical) resemble those of type II 10-30