真核生物的RNA聚合酶及其启动子Chapter_10_lecture

• Polymerase II makes
– Heterogeneous nuclear RNA (hnRNA)
– small nuclear RNA
• Polymerase III makes precursors to tRNAs, 5S rRNA and other small RNA
10-4
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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
• 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
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
RNA Polymerase Subunit Structures
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Polymerase II Structure
• For enzymes like eukaryotic RNA polymerases, can be difficult to tell:
– Which polypeptides copurify with polymerase activity
• 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
Chapter 10
Eukaryotic RNA Polymerases and Their Promoters
10.1 Multiple Forms of Eukaryotic RNA Polymerase
• There are at least two RNA polymerases operating in eukaryotic nuclei
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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
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
– 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
– 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
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Common Subunits
• There are five common subunits
– Rpb5 – Rpb6 – Rpb8 – b10 – Rpb12
• Little known about function • They are all found in all 3 polymerases
– 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