Eukaryotic RNA Polymerases and their Promoters Chapter 10
Multiple Forms of Eukaryotic RNA Polymerase – Early studies There are at least two RNA polymerases operating in eukaryotic nuclei One transcribes major ribosomal RNA (rRNA) 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 the nucleolus Depending on the organism each cell contains from several hundred to over 20,000 copies of rRNA gene. They are found in nucleolus and rest of the nuclear genes are present in nucleoplasm
Separation of the Three Nuclear Polymerases Eukaryotic nuclei contain three RNA polymerases Separated by ion-exchange chromatography RNA polymerase I found in nucleolus transcribes rRNA genes RNA polymerases II and III are found in the nucleoplasm - transcribes other kinds of RNA Robert Roeder and William Rutter in 1969 performed DEAE-Sephadex ion-exchange chromatography and studied three peaks of polymerase activity in order of their emergence from ion-exchange column.
Roles of 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
Polymerase Structure? Hard 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 For enzymes like eukaryotic RNA polymerases, can be difficult to tell
RNA Polymerase Subunit Structures Pierre Chambon and Rutter studied RNA polymerase II in great detail. In prokaryotes it was possible to separate the subunits and study what is required to reconstitute an eukaryotic nuclear polymerase from its separate subunits. This could not be performed in eukaryotic RNA polymerase. The other approach tried was mutating the subunits and determining what is required for what activity? Altogether 12 subunits have been found in yeasts and humans
Epitope tagging-Richard Young 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
Polymerase II Young - 10 subunits are placed in 3 groups: Core – (3 of the subunits) - related in structure and function to bacterial core subunits Common – (5 of the subunits) - found in all 3 nuclear RNA polymerases in yeast Nonessential subunits – (2 of the subunits) - conditionally dispensable for enzymatic activity
Core Subunits Three polypeptides - Rpb1, Rpb2, Rpb3 -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
Common Subunits There are five common subunits Rpb5 Rpb6 Rpb8 Rpb10 Rpb12 Little known about function They are all found in all 3 polymerases Suggests play roles fundamental in transcription They are all found in 3 polymerases in yeasts
Subunits Nonessential for Elongation Rpb4 and Rpb7 Dissociate fairly easily from polymerase 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 Rpb7 is an essential subunit - so must not be completely absent in the mutant
The Three-Dimensional Structure of RNA Polymerase II Structure of yeast polymerase II (pol II 4/7) - reveals a deep cleft that accepts a linear DNA template from one end to another Catalytic center lies at the bottom of the cleft and contains a Mg2+ ion Upper jaw – Rpb1+Rpb9 and lower jaw – Rpb5 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 It contains two Mg 2+ ions but one is lesser in concentration. Cleft has basic residues which bind the enzyme to acidic DNA template. When nucleic acids are present, the clamp region of the polymerase has shifted 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.
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
Position of Critical Elements in the Transcription Bubble Three loops of the transcription bubble are: - Rudder: initiating RNA- DNA dissociation Lid: maintains RNA-DNA dissociation Zipper: maintaining dissociation of template DNA Formation and maintenance of transcription bubble and dissociation of RNA-DNA hybrid. If the RNA-DNA hybrid extended farther than 9 bp, the rudder would be in the way. Rudder facilitate dissociation of hybrid.
Transcription mechanism 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 The active center of the enzyme lies at the end of pore 1. A polymerase can pause and then move backwards extruding the 3’ end of nascent RNA out of the enzyme.There is a pore on bottom of enzyme called pore 1 that might serve as exit point for RNA extrusion. Backtracks happen when nucleotide is misincorporated, thus exposing misincorporated nucleotide to removal by TFIIS which binds to the funnel at the other end of pore 1.
Class II promoters Class II Promoters - recognized by RNA polymerase II - are similar to prokaryotic promoters Considered to have two parts: Core promoter having 4 elements Upstream promoter element
Core Promoter Elements – 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
Other core elements - 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
Upstream promoter 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)
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
Three types of class III promoters Type I (5S rRNA) has 3 regions: Box A Short intermediate element Box C Type II (tRNA) has 2 regions: Box B Type III (nonclassical) resemble those of type II 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
Enhancers and Silencers These are position- and orientation-independent 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
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