Chapter 18 - Eukaryotic Transcription Flashcards
Differences from prokaryotic transcription
Chromatin must be relaxed before RNA polymerase can access the promoter
Requires many external initiation factors
General transcription factors
Multiple promoters and control elements
Multiple RNA polymerases responsible for transcription of different classes of genes
RNA Polymerase I Promotors
Transcribes rRNA genes from a single promoter type
Exists as a holoenzyme that is recruited to the promoter as a large complex by transcription factors
Core promoter is sufficient for initiation of transcription
core promoter
Surrounds the start point
Primarily GC rich
Efficiency is increased by interactions with upstream promoter (control) element
UBF
Required for high initiation frequency
Twists DNA to bring UPE and core promoter in close proximity to one another
Maintains open chromatin structure
Prevents H1 binding
SL1
Responsible for RNAP I recruitment
Binds to core promoter
Contains a TATA-binding protein
Three types of RNAP III promoters
Type 1
5S rRNA genes
Internal promoters located downstream of start
Type 1 and 2
tRNA genes
Internal promoters located downstream of start
Type 3
snRNA genes
Located upstream of start
Contains TATA box
Similar to RNAP II promoter
TFIIIB
Binds at start site
Its sole presence is sufficient for RNAP III to identify and bind start site
All RNAP III promoters require … to assist the binding of TFIIIB at the correct location
TFIIIC assembly factors
RNA polymerase II requires
general transcription factors to initiate transcription
RNAP II promoters are more diverse in their
structure than the bacterial promoter or the other eukaryotic RNAP promoters
TATA box
Common component of RNAP II promoters
The most important element for many RNAP II promoters
Similar in sequence to -10 consensus in bacteria
Often surrounded by GC rich sequences
BRE sequence
Located at approximately -30
Is the only upstream promoter element found at a relatively fixed position
initiator element
INR
covers transcription start site
downstream promoter element
DPE
common component of those RNAP II promoters that do not contain a TATA box
Each class of eukaryotic RNAP is assisted by a
positioning factor that contains TBP and other components
TATA-binding protein was originally identified as a
protein that binds to the TATA box in RNAP II promoters
TFIID
Positioning factor required by RNAP II
Also contains 14 subunits called TAFs
TBP associated factors
Multiple TFIID variants contain different combinations of TAFs
Different TFIID variants are tissue-specific
TBP
The positioning factor recognizes the promoter in different ways for different RNAPs
RNAP III
TFIIIB binds next to
TFIIIC
RNAP I
SL1 binds in conjunction with
UBF
RNAP II
TFIID is
solely responsible for binding
TBP binds to the
minor groove in DNA
nucleosome also bind in the
minor groove
upon binding, TBP bends the DNA
80 degrees
Three basic types of chromatin with respect to transcriptional activity
- Inactive gene with closed chromatin
- Potentially active gene with open chromatin and a bound RNAP
Poised gene
Basal apparatus is assembled but cannot transcribe without additional signal - Gene undergoing initiation in open chromatin
Active transcription begins
Transcription initiation complex steps
- TBP subunit of TFIID directs transcription factor to TATA box
- TFIIB binds
- TFIIF binds
- RNAP II is recruited to the promoter
- TFIIH binds
- TFIIE binds
TFIIB
Is recruited to the promoter along with RNAP II
TFIIF
Is recruited to the promoter along with RNAP II
Large subunit contains DNA helicase activity
Small subunit has some homology to bacterial sigma factor regions that bind core polymerase
RNAP II is recruited to the promoter
TFIIB binds near RNA exit site and may influence switch from abortive initiation to promoter escape
TFIIB also inserts into the active site of RNAP II and assists TFIID with stabilization of promoter melting
TFIIH
10 subunits, almost as large as RNAP II
Kinase that phosphorylates the CTD of RNAP II
Interacts with RNAP II downstream of start site
Involved in promoter escape
Involved in nucleotide excision repair pathways
TFIIE
Extends region covered by the apparatus to +30 degrees
TFIID binds to the INR via interactions with
TAFs
Some TATA-less promoters lack
unique transcription start sites
After the transcription initiation complex forms, TFIIH
hydrolyzes ATP to denature DNA at the transcription start site
RNAP II begins to make short unstable transcripts 4-5 nt in length
Similar to the abortive initiation events seen in bacterial initiation
Short transcripts are not base paired correctly
Promoter proofreading?
RNAP II must undergo conformational changes for
promoter clearance
promoter clearance
Ability of RNAP to release promoter and elongate transcript
Controlled by CTD and enhancers
Key determining factor of whether a gene is actually transcribed
Phosphorylation of CTD tail of RNAP II is required for
promoter and transcription factor release
Phosphorylation is facilitated by a kinase complex that includes TFIIH and Cdk9
TFIIH and Cdk9
TFIIH and Cdk9
TFIIH phosphorylates serines in the fifth position of each repeat
Cdk9 is also involved in cell cycle control
CTD is also involved in mRNA processing
Phosphorylated CTD serves as a recognition site for capping, tailing, and splicing enzymes
RNAP II changes conformation
Disengages from general transcription factors
Tightens interactions with DNA
Acquires new proteins that increase RNAP II processivity
Transcriptional regulators bind to enhancer regions to influence the assembly of the
general transcription factors and RNAP II to the gene control region
Enhancers are
cis-regulatory sequences located a variable distance from core promoter
Regulators that bind enhancers can be classified by their potential effect on transcription
activators and repressors
Other regulators called … also interact with activators and repressors
But do not usually directly bind DNA
coactivators and co-repressors
true activators
that bind specific DNA elements and the basal machinery at the promoter
chromatin remodeling activators
recruit chromatin modification enzymes and remodeling complexes
architectural modifying activators
bend DNA in order to bring factors bound apart on linear duplex into close proximity
mediator
A large protein complex that allows the transcriptional regulators, general transcription factors, and RNAP II to assemble at the promoter
Correctly positions TFIIH near the tail of RNAP II, which facilitates CTD phosphorylation
These new regulators act in three ways to facilitate elongation
- Recruit chromatin remodeling complexes to release chromatin that is blocking RNAP II movement
- Interacts with RNAP II via a coactivator to unpause enzyme
- Act as or recruit elongation factors
Elongation factors decrease the likelihood that RNAP will
dissociate from the DNA during elongation
Major function of elongation factors
is to help RNAP move through nucleosomes
Chromatin must be partially remodeled to facilitate transcription
Nucleosome sliding
Nucleosome removal
Replacement with histone variant nucleosomes
Histone modifications
Histone modification is an important part of
both transcription initiation and elongation
Initiation can be facilitated by
activators that recruit coactivators that contain histone modifying and chromatin remodeling enzymes
During elongation, nucleosomes ahead of RNAP are
acetylated, removed, and deposited behind the polymerase
Deposited nucleosomes are rapidly
deacetylated and methylated by polymerase-associated enzymes
FACT
FAcilitates Chromatin Transcription
Heterodimeric protein factor
Acts like a transcription elongation factor
Not part of RNA polymerase
Only associates during elongation
Helps facilitate H2A-H2B dimer release from octamers
FACT steps
FACT releases one H2A-H2B dimer from each octamer as RNA polymerase approaches
Remaining “hexosome” remains on DNA as RNA polymerase passes
After RNA polymerase passes, FACT adds H2A-H2B dimer back to “hexosome” to reform octamer
Chromatin structure is maintained
No FACT present
RNA polymerase and elongation factors peel some DNA from nucleosomes
Aided by supercoiling
DNA binding region of nucleosome is now accessible
Upstream DNA is looped and binds to exposed nucleosome
Nucleosome is captured by upstream DNA and transferred upstream and behind RNA polymerase
Eukaryotic transcription is thought to be … by default because of chromatin structure
off
Eukaryotic repressors are able to
both actively turn “off” a gene that has been activated and to further repress a gene that is already “off”
Eukaryotic repressors action
Prevent activator binding and action
Recruit histone modification and chromatin remodeling enzymes
