Transcript 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
•
•
•
This project is funded by a grant awarded under the President’s Community Based Job Training Grant as
implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
NCC is an equal opportunity employer and does not discriminate on the following basis:
against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
political affiliation or belief; and
against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in
the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
Disclaimer
• This workforce solution was funded by a grant awarded under the
President’s Community-Based Job Training Grants as implemented
by the U.S. Department of Labor’s Employment and Training
Administration. The solution was created by the grantee and does
not necessarily reflect the official position of the U.S. Department of
Labor. The Department of Labor makes no guarantees, warranties,
or assurances of any kind, express or implied, with respect to such
information, including any information on linked sites and including,
but not limited to, accuracy of the information or its completeness,
timeliness, usefulness, adequacy, continued availability, or
ownership. This solution is copyrighted by the institution that
created it. Internal use by an organization and/or personal use by
an individual for non-commercial purposes is permissible. All other
uses require the prior authorization of the copyright owner.