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

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

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

RNA Polymerase Subunit Structures

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 ’-, -, and -subunits• Both Rpb1 and ’-subunit binds DNA• Rpb2 and -subunit are at or near the

nucleotide-joining active site• Rpb3 does not resemble -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

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

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

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

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

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

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 A – Box B

• Type III (nonclassical) resemble those of type II

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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