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

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

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 10-2
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 10-3
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 • 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 blue • DNA nontemplate strand shown in green • RNA is shown in red 10-14
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 10-15
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 10-16
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 10-17
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 10-18
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 10-19
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 10-20