Lecture Molecular biology (Fifth Edition): Chapter 10 - Robert F. Weaver

In chapter 6 we learned that bacteria have only one RNA polymerase, which makes all three of the familiar RNA types: mRNA, rRNA, and tRNA. In this chapter we will see that three distinct RNA polymerases occur in the nuclei of eukaryotic cells. Each of these is responsible for transcribing a separate set of genes, and each recognizes a different kind of promoter.
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Lecture Molecular biology (Fifth Edition): Chapter 10 - Robert F. WeaverLecture PowerPoint to accompanyMolecular 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 10-2Separation 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 10-3 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 10-4RNA 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• Epitope tagging is a technique to help determine whether a polypeptide copurifies or is a subunit 10-6 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 10-7 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 10-8 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 10-9 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 10-10 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 10-11 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 10-12 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 10-13 Position of Nucleic Acids in the Transcription Bubble• DNA template strand is shown in bl ...