Transcript Inquiry into Life Twelfth Edition

Molecular Biology
Lecture 16
Chapter 10
Eukaryotic RNA
Polymerases and
Their Promoters
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Separation of the Three Nuclear
Polymerases
• Eukaryotic nuclei contain three RNA
polymerases
These can be separated by ion-exchange chromatography
10-2
Separation of the Three Nuclear
Polymerases
nucleoplasm
nucleolus
•RNA polymerase I found in nucleolus
–Location suggests in transcribes rRNA genes
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
• Technique to help determine whether a
polypeptide copurifies or is a subunit is
called epitope tagging
10-6
Epitope Tagging
• 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
10-7
10-8
Polymerase II
Original 10 subunits are placed in 3 groups:
• Core – related in structure and function to
bacterial core subunits
• Common – found in all 3 nuclear RNA
polymerases
• Nonessential subunits – conditionally
dispensable for enzymatic activity
10-9
Core Subunits
• Three polypeptides, Rpb1, Rpb2, Rpb3 are
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
– 2 monomers per holoenzyme
10-10
Common Subunits
• There are five common subunits
– Rpb5
– Rpb6
– Rpb8
– Rpb10
– Rpb12
• Little known about function
• They are found in all 3 polymerases
• Suggests play roles fundamental in
transcription
10-11
Subunits Nonessential for
Elongation
• Rpb4 and Rpb7
–
–
–
–
–
Dissociate fairly easily from polymerase
Found in substoichiometric quantities
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, so must not be
completely absent in the mutant
10-12
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 carboxylterminal domain (CTD) which is 7aa-peptide repeated over and
over. Enzyme with IIa binds to the promoter
– Converts to IIo by phosphorylating 2 ser in the repeating heptad
of the CTD. Enzyme with IIo is involved in transcript elongation
10-13
The Three-Dimensional
Structure of RNA Polymerase II
• Structure of yeast polymerase II (specifically pol II 4/7)
at atomic resolution reveals a deep cleft that accepts a
linear DNA template from one end to another
10-14
• Catalytic center lies at the bottom of the cleft and contains
a Mg2+ ion
• A second Mg2+ ion present in low concentrations
• 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
10-15
Promoters
• Three eukaryotic RNA polymerases have:
– Different structures
– Transcribe different classes of genes
• Expect that the 3 polymerases would
recognize different promoters
10-16
Class II Promoters
• Promoters recognized
by RNA polymerase II
(class II promoters)
are similar to
prokaryotic promoters
• Considered to have
two parts:
– Core promoter having
4 elements
– Upstream promoter
element
10-17
10-18
Core Promoter Elements – TATA
Box
• 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
10-19
Core Promoter Elements
• In addition to TATA box, core promoters are:
– 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
10-20
Upstream Elements
• 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)
• Upstream promoter elements can be orientationindependent, yet are relatively positiondependent
10-21
Upstream Elements
10-22
Enhancers and Silencers
• These are position- and orientationindependent 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
10-23
10-24
10-25
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
10-26
RNA polymerase 1 promoters
10-27
10-28
Class III Promoters
• 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
10-29
Promoters of Some
Polymerase III Genes
• 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
10-30