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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. | Molecular Biology Fifth 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. 10.1 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- 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- Roles of the Three RNA Polymerases Polymerase I makes large rRNA precursor Polymerase II . | Molecular Biology Fifth 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. 10.1 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- 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- 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- RNA Polymerase Subunit Structures 10- 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- 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- 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 .