Lecture 17 Flashcards
what is necessary for recognition of promoters in eukaryotes
transcription factors
GTF (general transcription factors) are
equivalent to sigma factor of bacteria
TFII”x”
RNAPII
TFIII”X”
RNAP III
RNAP I and III rely on
a small number of factors that recognize a small number of promoters
RNA POL I products
18S, 25S, 5.8S rRNAs
RNA Pol I promoter elements
UCE, core sequence
RNA Pol I promoter elements
UCE, core sequence
All thre types of RNA pol use a TF called
TBP - TATA binding protein
TATA box
5-TATAAA sequence near -30 position ‘
- TBP is used for transription initation of all genes (presenting or not TATA box)
TAFs
TAFs (TBP associated factors): proteins that recruit TBP to genes that lack TATA box.
RNA polymerase I
Transcription of rRNA genes (rRNA precursor)
- RNA pol I “functions” within nucleolus
- 45S RNA is synthesized and tgen processed to 5.8S, 18S and 28S
UBF
upstream binding factor
SL1
selectivity factor (complex of TBP and three TAFs)
SL1 binds UBF and Pol I
RNA pol III products
tRNA
5S rRNA
7SL RNA
RNA Pol III transcription of
tRNAs, 5S rRNA, small ncRNA
tRNA requirement
TFIIIB and TFIIIC
5S rRNA requirement
TFIIIB, TFIIIC, TFIIIA
other small RNAs require
other speciffic factors in addition to TFIIIB - like
TFIIIB includes
TBP (positioning factor)
termination of transcription
RNA Pol I and III
RNA Pol I and III terminate (like prokaryotes) at specific terminators
RNA POL I terminator
terminator site located downstream from the pre-rRNA sequence; requires additional proteins (termination factors that recognize terminator)
RNA Pol III termination
terminates transcripriton at T rich sequences in the DNA template kocated a short distance from the 3’ end of the mature RNA assisted by just a few protein factors
RNAP II transcribes
mRNAs
MicroRNAs
some ncRNAs
RNAP II comes in many variteties but share certain sequence features
RNAP II promoters sometimes include
a TFIIB recignition element (BRE) and the downstream promoter element (DPE)
RNAP II promoters require several factors for the assembly of the basal transcription apparatus:
RNAP II and TFIIA, B, D, E, F, H, and J
where does the basal transcription apparatus assemble
at the core promoter
All RNA PolII promoters are
molecular and composed of a few elements (cis acting sites) that will be bound by TFIID in a sequence specific manner
25% of human genes
contain TATA box - and upstream element usually present around -30
RNAP II core promoter contains _ around the start point + atleast one more element
initator (Inr)
- TATA box sequence
or -
TATA less, promoters must contain atleast one other element downstream from Inr
1. DPE - downstream promoter element
2. DCE - downstream core element
builidng of the initiation complex is sequential and
starts with TFIID
TFIID is composed of
14 subunits
TAFs - TBP associated factors
- different TAFS have different affinities for different sequence elements that may be oresent within different core promoters
TBP
- TATA binding protein binds to promoters (ONLY at TATA box when present)
TFIID binds to
Inr in TATA less promoters, first via TAF
other TAF or TAFs, depending on the promoter bind to downstream elements
initiation complex
once TFIID is bound the assembly of the complex is based on mostly protein-protein interactions
- TFIIA and TFIIB join after TFIID
- RNAP II joins witg TFIIF
- TFIIE and TFIIH (10 subunits) and TFIIJ enter the complex next
- TFIIH initiates formation of open complex
- CTD (carboxy terminal domain) of RNA POL II is phosphorylated by TFIIH
- Factors are released except TFIIH
transition from initation to elongation depends on
TFIIH
TFIIH
very large multi subunit protein with several enzymatic activities and functions
Functions of TFIIH
- helicase activity
- kinase activity
- *helicase activity
TFIIH
helicase activity
responsible for initial melting of the DNA at the promoter to form an open complex
Two subunits (XPB and XPD) are involved in TCR (transcription coupled repair)
kinase activity - TFIIH
phosphorylation of a CTD (carboxyl terminal domain) of the RNA pol II tail
CDT
multiple repeats of the seven amino acid sequence
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
CTD during initaiton
not phosphorylated
TFIIH phosphorylates
every serine 2 and 5 within CDT
RNAP II loses affinity for
GTFs and clears promoter
beginning of elongation is the
act of promoter clearance
the phosphorylation state of CTD determines
what proteins/protein complexes interact with RNAP II
elongation, transcription modification and transcript cleavage are
all dependen ton the modification state of CTD
Transcription at Pol II promoters
- Pol II recruited DNA by transcription factors
- Formation of transcription bubble
- phosphorylation of CTD during initation
- Elongation
- Dephosphorylation of CTD; transcription terminates
transcription initation in vivo requires the mediator complex
mediator: intermediary between specific transcription factors bound at upstream promoter elements and enhancers, and the POL II complex and general TF bound at the core promoter
activators
bind to genes at sites known as enhancers and speed the rate of transcription
repressors
bind to selected sets of genes at sites known as silencers and thus slow transcription
coactivators
adapter molecules integrate singals from activators and perhaps repressors
Basal transcription factors
in response to injunctions from activators, these factors position RNA polymerase at the start of transcription and initate the transcription process
termination of transcription RNA POL II
- Pol II termination does not occur at a conserved site or at a constant distance from the 3’ end of mature RNAs
- mammals: takes place anywhere from a few kb pairs to several kb pairs downstream from the 3’ end of the mature transcript
- Polyadenylation signal seqeuence (AAUAAA) is present in the pimary transcript (and directly encoded by the DNA)
- Factors responsible for cleabage of the primary transcript bind to the AAUAAAA sequence, resulting in cleavage somewhat downstream from that position
