Download Eukaryotic RNA Polymerases and their Promoters

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 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
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 RNADNA dissociation
– Lid: maintains RNADNA 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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